TW201427685A - Methods of using FIX polypeptides - Google Patents

Abstract

The present invention provides methods of administering long-acting Factor IX; methods of administering long-acting, chimeric and hybrid polypeptides comprising Factor IX; and methods of producing such chimeric and hybrid polypeptides using cells.

Description

Application of FIX polypeptide

The present invention generally relates to the field of therapeutic agents for use in hemostatic disorders.

Hemophilia B (also known as Christmas disease) is one of the most common hereditary bleeding disorders in the world. It results in reduced blood coagulation activity in vivo and in vitro and requires extensive medical monitoring during the lifetime of the affected individual. In the absence of intervention, the afflicted individual will suffer from spontaneous joint bleeding, which produces severe pain and debilitating inactivity; bleeding into the muscles can cause blood to accumulate in their tissues; if not treated immediately, spontaneous throat And neck bleeding can cause asphyxia; kidney bleeding; and severe bleeding after surgery, minor accidental injury or tooth extraction is also common.

Normal in vivo coagulation requires at least serine protease II (prothrombin), VII, IX, X and XI (soluble plasma proteins); cofactors, including transmembrane protein tissue factor and plasma protein factor V and VIII Fibrinogen, transglutaminase factor XIII, phospholipids (including activated platelets), and calcium. Other proteins, including kallikrein, high molecular weight kininogen, and factor XII, are required for some in vitro coagulation tests and can function in vivo under pathological conditions.

In hemophilia, blood clotting is lacking in certain plasma blood Interference with condensation factor. Hemophilia B is caused by a deficiency in Factor IX caused by a defective molecule that is reduced in synthesis or reduced in activity of Factor IX protein. In the absence of effective control, recurrent joint hemorrhage can lead to the development of progressive and disabling joint disease and lead to poor quality of life (Giangrande P., Expert Opin Pharmacother. 2005; 6: 1517-24). Hemophilia is treated by replacing the lost coagulation factor with a concentration of factor IX exogenous factor concentrate. However, as described below, it is technically difficult to produce such a concentrate from blood.

Purification of factor IX from plasma (plasma-derived factor IX; pdFIX) almost only active factor IX. However, this purification of Factor IX from plasma is extremely difficult because Factor IX is only present in plasma at low concentrations (5 ug/mL). Andersson, Thrombosis Research 7:451 459 (1975). In addition, purification from blood requires removal of infectious agents (such as HIV and HCV) or inactivation of infectious agents. In addition, pdFIX has a short half-life and therefore requires frequent administration, resulting in reduced compliance with prophylactic treatment. Recombinant Factor IX (rFIX) is also available, but suffers from shorter half-lives and requires frequent dosing as with pdFIX (eg, 2-3 times per week for control). rFIX also has a lower incremental recovery (K value) compared to pdFIX, making it necessary to use a higher dose than the dose of pdFIX. rFIX.

Reduced mortality, prevention of joint damage, and improved quality of life have been attributed to the development of plasma-derived factor IX and recombinant factor IX. Prolonging protection from bleeding will represent another key advance in the treatment of subjects with hemophilia B. However, to date, products that allow long-term protection have not been developed. The relatively short half-life of currently available FIX products requires frequent intravenous injections (2-3 times per week), thereby reducing compliance with control. See White GC et al, Thrombosis and Haemostasis 78: 261-5 :1997. In children, it is often necessary to use a central venous access device (CVAD), which further exposes the child to the risk of infection and thrombosis. See Hacker MR et al, Hemophilia , 10: 134-46 (2004). One of the serious potential risks associated with rFIX treatment is the production of inhibitors (neutralizing antibodies) that typically occur within the first 50 exposure days (ED). Inhibitors pose challenges to establishing hemostasis and can be associated with allergies. See Chitlur M. et al., Hemophilia , 15: 1027-31 (2009). Therefore, there is still a need for improved methods of treating hemophilia caused by a deficiency in the treatment of Factor IX that are more tolerable and more effective than current therapies.

The present invention provides a method of administering a long-acting Factor IX (FIX) polypeptide to a human subject in need thereof, comprising administering to the subject about 10 IU/kg at a dosing interval of about one week or more. The long acting FIX polypeptide is dosed to a dose of about 200 IU/kg. In one embodiment, the dosage of the long acting FIX polypeptide is from about 10 IU/kg to about 50 IU/kg, about 10 IU/kg. Up to about 100 IU/kg, from about 25 IU/kg to about 50 IU/kg, from about 25 IU/kg to about 75 IU/kg, from about 25 IU/kg to about 100 IU/kg, from about 25 IU/kg to about 125 IU/kg, about 25 IU/kg To about 150 IU/kg, from about 50 IU/kg to about 100 IU/kg, from about 50 IU/kg to about 150 IU/kg, from about 100 IU/kg to about 150 IU/kg, from about 150 IU/kg to about 200 IU/kg, or any combination thereof. In another embodiment, the dose of the long acting FIX polypeptide is used to control one or more bleeding events. In one example, the dose to control one or more bleeding conditions is about 50 IU/kg. In another example, the annual bleeding rate of a bleeding event following administration of the long acting FIX polypeptide is less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, or less than 10.

In another embodiment, the dosage of the long acting FIX polypeptide is One or more bleeding events are prevented at individualized intervals. In one example, the dosage of the long acting FIX polypeptide is about 100 IU/kg. In another example, the annual bleeding rate of a bleeding event following administration of the long acting FIX polypeptide is less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, or less than 9. In other embodiments, the dose of the long acting FIX polypeptide is used to treat one or more bleeding events as needed. In one example, the annual bleeding rate of the bleeding event after administration of the long acting FIX polypeptide is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19. Less than 20, less than 21, less than 22, less than 23, less than 24, less than 25 or less than 26. In other embodiments, the dosage of the long acting FIX polypeptide is for perioperative management of a bleeding disorder.

In some embodiments, from about once a week to about one month The dose is administered at a frequency of one administration. In one aspect, the frequency of administration is about once a week, once in about two weeks, twice in about one month, once in about three weeks, once in about four weeks, or once in about one month. In another aspect, the frequency of administration is about once a week. In other aspects, the frequency of administration is once in about two weeks or twice in about one month.

In some embodiments, the dosing interval is about every 5 days, about Every 6 days, about every 7 days, about every 8 days, about every 9 days, about every 10 days, about every 11 days, about every 12 days, about every 13 days, about every 14 days, about every 15 days, about every Every 16 days, about every 17 days, about every 18 days, about every 19 days, about every 20 days or about every 21 days. In other embodiments, the dosing interval is every 7 to 14 days.

The T _{1/2 β} (activity) of the long-acting FIX polypeptide can be at least about 40 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least About 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, or at least about 190 hours. In a particular example, T _{1/2 β} (activity) is from about 40 hours to about 193 hours. In another example, the average of T _{1/2 β} (activity) is about 82 hours.

The T _{1/2 β} (antigen) of the long-acting FIX polypeptide can also be at least about 60 hours, at least about 80 hours, at least about 100 hours, at least about 120 hours, at least about 140 hours, at least about 160 hours, at least about 180 hours, At least about 200 hours, at least about 220 hours, at least about 240 hours, at least about 260 hours, at least about 280 hours, at least about 300 hours, at least about 320 hours, at least about 340 hours, at least about 360 hours, or at least about 370 hours. In a particular embodiment, the T _{1/2 β} (antigen) is from about 63 hours to about 372 hours.

In some embodiments, after administration, in the subject The plasma trough content of the long acting FIX polypeptide is maintained at about 1% above baseline. In other embodiments, the plasma trough content of the long-acting FIX polypeptide in the subject is maintained between about 1% and about 5%, between about 1% and about 6%, between about 1% and about 7% above the baseline. Between, between about 1% and about 8%, between about 1% and about 9%, between about 1% and about 10%, between about 1% and about 11%, between about 1% and about 12% Between about 1% and about 13%, between about 1% and about 14%, between about 1% and about 15%.

In one embodiment of the method of the invention, the dosage is about 50 IU/kg, and the dosing interval is about 7 days. In another embodiment, the dosage is about 100 IU/kg and the dosing interval is at least about 14 days. In other embodiments, the dosage is about 150 IU/kg and the dosing interval is at least about 21 days.

Long-acting FIX polypeptides can be comprised of FIX polypeptides and FcRn binding A chimeric polypeptide of the conjugate. In one embodiment, the FcRn binding partner comprises an Fc region. In another embodiment, the long acting FIX polypeptide further comprises a second FcRn binding partner. In other embodiments, the second FcRn binding partner comprises a second Fc region. In a specific embodiment, the long acting FIX polypeptide is a FIX monomer dimer hybrid.

In some embodiments, the subject needs to control or prevent Blood or bleeding events. In other embodiments, the subject needs to control or prevent bleeding in the following: small blood loss, joint blood, superficial muscle loss, soft Tissue loss of blood, moderate blood loss, intramuscular or soft tissue loss with anatomy, mucous membrane loss, hematuria, massive blood loss, pharyngeal blood loss, posterior pharyngeal blood loss, retroperitoneal blood loss, central nervous system blood loss, abrasion, incision, scratch, joint Blood loss, nosebleeds, oral bleeding, bleeding gums, intracranial hemorrhage, intraperitoneal hemorrhage, a small amount of spontaneous blood loss, bleeding after major trauma, moderate skin abrasions, or spontaneous blood loss to the joints, muscles, internal organs or brain.

In one embodiment, the subject requires a perioperative tube Reason. In another embodiment, the subject needs to manage bleeding associated with surgery or tooth extraction. In other embodiments, the subject will experience, is undergoing, or has undergone major surgery. In one aspect, major surgery is orthopedic surgery, large-area oral surgery, urinary surgery, or hernia surgery. In another aspect, the orthopedic procedure is to replace the knee, hip or other major joint.

In some embodiments, the subject is in need of treatment for bleeding disorders Symptoms, such as joint blood, muscle bleeding, oral bleeding, blood loss, blood loss to the muscles, oral blood loss, trauma, head trauma, gastrointestinal bleeding, intracranial blood loss, intra-abdominal blood loss, intrathoracic blood loss, fracture, central nervous system bleeding Hemorrhage in the posterior pharyngeal space, hemorrhage in the retroperitoneal space, or hemorrhage in the iliopsoas. In other embodiments, the subject requires surgical management, perioperative management, or surgical treatment, such as surgery for minor surgery, major surgery, extraction, tonsillectomy, inguinal hernia, synovectomy, total knee replacement, Craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery or joint replacement surgery.

In some embodiments, the dosage is administered intravenously or subcutaneously.

The invention also includes an estimate for individualizing patients A method of administering information to rFIXFc, the method comprising: (a) receiving at least one of patient information and information on a desired treatment result by a computer-based system comprising the rFIXFc population pharmacokinetic (popPK) model of Example 6 and a Bayesian estimation program (b) using the popPK model, the Bayesian estimation program and the received information, calculating the individualized rFIXFc administration information by the computer-based system, and (c) outputting the individualized administration by the computer-based system News.

In some embodiments, the method also includes output based on (c) The individualized dosing information selects the dosing regimen and doses rFIXFc to the patient according to the selected dosing regimen.

In some embodiments, the information to be treated is given The degree of plasma FIX activity is increased after the drug and the output information is the dose for acute treatment.

In some embodiments, the desired treatment outcome information is the desired dosing interval and the output information is the dose for control.

In some embodiments, the information to be treated is the desired dose and the output information is the interval for control.

The invention also includes a method for estimating a rFIXFc dosing regimen based on a median popPK, the method comprising: (a) receiving patient information and information about a desired treatment result by a computer-based system comprising the rFIXFc popPK model of Example 6 and a Bayesian estimation program At least one of (b) using the popPK model, the Bayesian estimation program, and the received information, calculating the median rFIXFc PK information by the computer-based system, and (c) borrowing The median PK information is output by the computer based system.

In some embodiments, the method also includes output based on (c) The median PK information selects the dosing regimen and doses rFIXFc to the patient according to the selected dosing regimen.

The invention also includes an estimate of individual patient rFIXFc PK The method comprises: (a) receiving individual rFIXFc PK information by a computer-based system comprising the rFIXFc population pharmacokinetic (popPK) model of Example 6 and a Bayesian estimation program, (b) using a popPK model, a Bayesian estimation program And receiving the information, estimating the individualized patient rFIXFc PK information by the computer based system, and (c) outputting the individualized patient PK information by the computer based system.

In some embodiments, the method also includes output based on (c) The individualized patient PK information selects the dosing regimen and the patient is administered rFIXFc according to the selected regimen.

In some embodiments, (a) further includes The brain system receives patient information.

In some embodiments, the patient information is age or weight.

Other embodiments include a storage that is executed by a processor A computer readable storage medium that causes the processor to execute instructions of any of the above methods.

Other embodiments include a processor and a memory A system having stored thereon instructions that, when executed by the processor, cause the processor to perform any of the above methods.

1900‧‧‧ computer system

1902‧‧‧Display interface

1904‧‧‧ Processor

1906‧‧‧Communication infrastructure

1908‧‧‧ main memory

1910‧‧‧Auxiliary memory

1912‧‧‧hard disk drive

1914‧‧‧Removable storage drive

1916‧‧‧Removable storage unit

1920‧‧‧ interface

1922‧‧‧Removable storage unit

1924‧‧‧Internet interface

1926‧‧‧Communication Path

1930‧‧‧Display unit

Figure 1 shows the study design and CONSORT diagram. Exceeding work Efficacy data collected during the period of efficacy was not included in the efficacy analysis. * The PK subgroup was dosed with rFIX, followed by PK evaluation and elution (greater than or equal to 5 days) prior to rFIXFc administration for PK evaluation. rFIX sampling was performed as follows: 10 (±2) minutes, 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 before injection. (±2) hours, 72 (±3) hours, and 96 (±3) hours (4 days). rFIXFc sampling was performed as follows: 10 (±2) minutes, 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 before injection. (±2) hours, 96 (±3) hours (4 days), 144 (±3) hours (6 days), 168 (±3) hours (7 days), 192 (±3) hours (8 days) and 240 (±3) hours (10 days). Infusion was performed within 10 minutes. Blood samples from each subject were collected over 96 hours. Repeated PK assessment of rFIXFc was also performed at week 26. ED = exposure day; PK = pharmacokinetics.

Figure 2 shows an overview of continuous administration and PK sampling of team 1.

Figure 3A shows subjects in teams 1, 2, and 3, respectively. A graph of the median annual bleeding rate (ABR). A bar chart showing the overall, spontaneous, and traumatic bleeding of each team.

Figure 3B shows an overview of the annual bleeding rate for the subgroups. All analyses were consistent with primary efficacy analysis, showing a reduction in bleeding rates across all subgroups. Team 1 is the only team that has a sufficient number of patients to graphically display the subgroup analysis.

Figure 4 shows the use in the continuous pharmacokinetic group (team 1) Baseline corrected mean FIX activity (IU/dL) over time in a single-stage coagulation assay (log scale) obtained from rFIX and rFIXFc treated individuals.

Figure 5 shows three compartments for predicting the population PK of rFIXFc A diagram of a room model. CL, clearance rate; V, volume of distribution; Q, clearance rate between compartments.

Figure 6 shows the baseline (week 1) and repeated PK (week 26) Clearance rate (CL) and V1 estimate (black line indicates the average of no large changes between the two cases).

Figure 7 shows individual PK parameters relative to body weight (BW).

Figure 8 shows FIX live predicted by population or individual PK models The fit is better than the observed FIX activity.

Figure 9 shows a population of 50 IU/kg or 100 IU/kg dose Visual predictive check (VPC) map of the PK model. The red and black lines represent the 10th, 50th and 90th percentiles of the simulated (red) and observed (black) data, respectively.

Figure 10 shows the population PK model verified using valley/peak records. R2 = 0.9857, P < 0.001.

Figure 11 shows a map of the rFIXFc pediatric study design.

Figure 12 shows the observed and predicted perioperative FIX activity Representative map.

Figure 13 shows population simulation of steady-state FIX activity time profiles (5th - 95th percentile).

Figure 14 shows a simulation of steady state FIX activity versus time profile, It compares the 5th to 95th percentiles of the group once a week 50 IU/kg vs. 4000 IU and 100 IU/kg per 10 days versus 8000 IU.

Figure 15 shows the output of the proposed evaluation for individual PKs.

Figure 16 shows the proposed output for an individualized dosing regimen for paroxysmal therapy.

Figure 17 shows the output of the proposed dosing regimen selection without individualized PK assessment.

Figure 18 shows another output of a dosing plan selection proposal without an individualized PK assessment.

Figure 19 shows an example computer system that can be used in an embodiment.

Figure 20 shows a graph plotting the predicted total bleeding versus time at a level of 1% FIX activity.

Figure 21 shows a graph plotting the predicted total bleeding versus time at a level of 5% FIX activity.

The present invention provides a drug delivery interval that is longer than the dosing interval, dosing frequency, and/or pharmacokinetic parameters that may be achieved with currently known Factor IX products, longer dosing frequency, and/or improved drug delivery. Kinetic parameters, a method of treating FIX deficiency ( eg , hemophilia B) with FIX. The invention also provides improved long acting FIX polypeptides, FIX chimeric polynucleotides, and methods of manufacture.

I. Definition

The term "about" is used herein to mean approximating, roughly, approximately, or about. When the term "about" is used in conjunction with a numerical range, it is modified by extending the boundary above and below the stated value. In general, the term "about" is used herein to modify a value above and below the value by 10% up or down (increased or decreased).

The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymeric compound comprising covalently linked amino acid residues.

The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound comprising covalently linked nucleotide residues. The polynucleotide may be single or double stranded DNA, cDNA, RNA, vector, plastid, phage or virus. The polynucleotides include the polynucleotides encoding the polypeptides of Table 19 in Table 18 (see Table 18). Polynucleotides also include fragments of the polynucleotides of Table 18, such as a polynucleus encoding a fragment of the polypeptide of Table 19 (such as Factor IX, Fc, signal sequence, propeptide, 6His, and other fragments of the polypeptide of Table 19). Glycoside fragment.

The term "administering" as used herein means to administer or administer a pharmaceutically acceptable long-acting FIX polypeptide of the invention to a subject via a pharmaceutically acceptable route. Examples of routes of administration include, but are not limited to, intravenous routes such as those achieved via central venous access, such as intravenous injections and intravenous infusions. Other routes of administration include subcutaneous, intramuscular, oral, nasal, and transpulmonary administration, preferably subcutaneous administration. The long acting FIX polypeptide (FIX chimeric or hybrid protein) can be administered as part of a pharmaceutical composition comprising at least one excipient. Advantages of the present invention include: program compliance change Good; breakthrough bleeding reduction; increased protection against joint bleeding; joint damage prevention; reduced rickets; reduced mortality; long-term defense against bleeding; reduced thrombosis events; and improved quality of life.

The term "chimeric polypeptide" as used herein means a polypeptide comprising at least two polypeptides (or portions thereof, such as subsequences or peptides) from different sources. A chimeric polypeptide can include two, three, four, five, six, seven or more polypeptides or portions thereof from different sources, such as different genes, different cDNAs, or different animals or other species. A chimeric polypeptide can include one or more linkers that join different polypeptides or portions thereof. Thus, within a single chimeric polypeptide, the polypeptide or portion thereof can be joined directly or it can be joined indirectly via a linker or in both direct and indirect ways. Chimeric polypeptides can include other peptides (such as signal sequences) and sequences (such as 6His and FLAG) that facilitate protein purification or detection. Furthermore, the chimeric polypeptide may have an amino acid or peptide addition at the N-terminus and/or C-terminus. An exemplary chimeric polypeptide of the invention is a Factor IX-FcRn BP chimeric polypeptide, such as a Factor IX-Fc chimeric polypeptide, such as FIXFc in Figure 1, SEQ ID NO: 2 (Table 18), with or without its signal sequence And propeptide.

The terms "long-lasting" and "durable" are used interchangeably herein. In one embodiment, the term "long-term" or "persistent" instructions caused by the administration of the "long-term" FIX polypeptides FIX activity than the wild type FIX (e.g. BENEFIX ^® or plasma-derived FIX ( "pdFIX")) is FIX activity is long-lasting. The "longer" FIX activity can be measured by any known method in the art, such as aPTT analysis, color analysis, ROTEM, TGA, and the like. In one embodiment, "longer" FIX activity can be indicated by T _{1/2 β} (activity). In another embodiment, "longer" FIX activity can be indicated by the amount of FIX antigen present in the plasma, such as by T _{1/2 β} (antigen). In other embodiments, as compared to wild-type FIX polypeptide (i.e., by the SEQ ID NO: 2 amino acids 1-415 of the polypeptide composition, i.e. BENEFIX ^® or pdFIX), long-acting or persistent stage coagulation FIX polypeptides The association has a longer function, such as a longer active period.

Factor IX coagulation activity is expressed in International Units (IU). The Factor IX activity of one IU corresponds approximately to the amount of Factor IX in one milliliter of normal human plasma. Several assays can be used to measure factor IX activity, including single-stage coagulation analysis (activated partial clotting hormone time; aPTT), thrombin generation time (TGA), and rotational thrombosis (ROTEM ^® ).

The term "frozen" as used herein in connection with the formulation of the present invention Lyophilisate (which may also be used interchangeably with "lyophilizate") means a formulation made by the freeze-drying method known per se in the art. The solvent (eg, water) is removed by freezing after sublimation under vacuum and desorbing residual water at elevated temperatures. In the pharmaceutical field, lyophilizates typically have from about 0.1% to 5% (w/w) residual moisture and are present in the form of powders or stable solid cakes. The lyophilizate is characterized by rapid dissolution after the addition of the healing medium.

The term "reconstituted formulation" as used herein means a formulation that is lyophilized and redissolved by the addition of a diluent. The diluent may contain, without limitation, water for injection (WFI), water for sterilizing injection (BWFI), sodium chloride solution ( for example, 0.9% (w/v) NaCl), glucose solution (for example, 5% glucose), and interfacial activity. A solution of the agent ( eg, 0.01% polysorbate 20 or polysorbate 80), a pH buffer solution (eg, a phosphate buffer solution), and combinations thereof.

As used herein, "dosing interval" means to the subject The length of time between the multiple doses administered. Therefore, the dosing interval can be indicated as a range. In the method of the invention using a chimeric FIX-FcRnBP such as chimeric FIX-Fc, the dosing interval can be compared to an equal amount (in IU/kg) of the factor IX without FcRn BP (eg, Fc portion) (ie, by The polypeptide consisting of the FIX) requires a dosing interval of at least about 1.5 to 8 times longer. When administered, for example, a Factor IX-Fc chimeric polypeptide (or hybrid) of the present invention, the administration interval can be compared to an equivalent amount of the Factor IX without FcRn BP (e.g., Fc portion) (i.e., a polypeptide consisting of the Factor IX) The required dosing interval is at least about 1.5 times longer. The dosing interval can be at least about 1.5 to 8 times longer than the dosing interval required for an equal amount of the Factor IX (or polypeptide consisting of the Factor IX), such as the Fc portion.

The term "dosing frequency" as used herein refers to a given The frequency of administration of the dose of the long-acting FIX polypeptide over time. The frequency of dosing can be indicated as the number of doses per given time, such as once a week or once every two weeks.

The term "bleeding event" as used herein is given in a standardized definition: a bleeding event begins with the first bleeding sign and ends at 72 hours after the last treatment bleeding, and any bleeding symptoms or differences between the same locations within the 72 hours are less than An injection equal to or greater than 72 hours is considered the same bleeding event. See Blanchette V. (2006) Haemophilia 12 : 124-7. As used herein, any injection of a therapeutic bleeding event performed more than 72 hours after the previous bleeding event is considered to be the first injection to treat a new bleeding event at the same location. Similarly, any bleeding at different locations is considered an independent bleeding event regardless of the time of the last injection.

As used herein, the term "controls one or more bleeding events Or "control" means administering a Factor IX polypeptide to a subject in multiple doses over a period of time to increase the level of Factor IX activity in the subject's plasma. In one embodiment, "controlling one or more bleeding events" indicates the use of a long-acting FIX polypeptide to prevent or inhibit the occurrence of one or more spontaneous or uncontrolled bleeding or bleeding events, or to reduce one or more of the spontaneous or uncontrollable events. The frequency of bleeding or bleeding events. In another embodiment, the increased degree of FIX activity is sufficient to reduce the incidence of spontaneous bleeding or to prevent bleeding without foreseeable injury. Prophylactic treatment reduces or prevents bleeding events, such as those described in on-demand treatment. As discussed under "Dosing intervals," prophylactic treatment can be individualized, for example, to counteract inter-subject variability.

The term "about once a week" as used herein means approximation The value and "about once a week" may include ± two days every seven days, that is, every five days to every nine days. Therefore, the frequency of "once a week" can be every five days, every six days, every seven days, every eight days or every nine days.

The term "individualized interval control" as used herein means Use of a long-acting FIX polypeptide at an individualized dosing interval or frequency to prevent or inhibit the occurrence of one or more spontaneous and/or uncontrollable bleeding or bleeding events, or to reduce one or more spontaneous and/or uncontrollable bleeding or bleeding events The frequency. In one embodiment, the "individualized interval" includes ±3 days per 10 days, that is, every 7 days to every 13 days. Therefore, the frequency of administration of "individualized interval control" can be every 7 days, every 8 days, every 9 days, every 10 days, every 11th, every 12th or every 13th.

The term "on-demand treatment" as used herein means intended Treatment that takes place over a short period of time and responds to an existing condition, such as a bleeding event or a perceived short-term need, such as a planned surgery. "On-demand treatment" can be used interchangeably with "episodic" treatment. Conditions that may require on-demand treatment include bleeding events, joint blood, muscle bleeding, oral bleeding, blood loss, blood loss to the muscles, oral blood loss, trauma, head trauma, gastrointestinal bleeding, intracranial blood loss, intra-abdominal blood loss, chest Internal blood loss, fracture, central nervous system hemorrhage, hemorrhage in the posterior pharyngeal space, hemorrhage in the retroperitoneal space, or hemorrhage in the iliopsoas. It also includes bleeding events other than these conditions. Subjects may require surgery, perioperative management, or surgery. These procedures include minor surgery, major surgery, extraction, tonsillectomy, other dental surgery/thoracic surgery, inguinal hernia incision, synovectomy, total knee replacement, other joint replacement, craniotomy, osteosynthesis, Trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery. It also includes surgery other than these operations.

Other conditions that may require on-demand treatment include a small amount of loss Blood, joint blood, superficial muscle loss, soft tissue blood loss, moderate blood loss, anatomical intramuscular or soft tissue loss of blood, mucosal blood loss, hematuria, massive blood loss, pharyngeal blood loss, posterior pharyngeal blood loss, retroperitoneal blood loss, central nervous system Systemic blood loss, abrasion, incision, scratch, joint blood loss, nosebleed, oral bleeding, bleeding gums, intracranial hemorrhage, intraperitoneal hemorrhage, a small amount of spontaneous blood loss, bleeding after major trauma, moderate skin abrasions or joints, muscles, internal organs Or spontaneous blood loss in the brain. Other causes of on-demand treatment include perioperative management surgery or extraction, major surgery, large-scale oral surgery, and secretion Urine surgery, hernia surgery, orthopedic surgery, such as replacement of the knee, hip or other major joints.

The term "treatment" as used herein means to improve or alleviate Includes, but is not limited to, one or more of the bleeding disorders or conditions of hemophilia B. In one embodiment, "treating" a bleeding disease or condition includes preventing one or more symptoms of a bleeding disease or condition. In the case of a bleeding disease or condition caused by a lack of FIX (eg, low baseline FIX activity), the term "treatment" means FIX replacement therapy. By administering a long-acting FIX polypeptide to a subject, the subject can achieve and/or maintain a plasma FIX valer activity level of about 1 IU/dl or greater than 1 IU/dl. In other embodiments, "treating" means reducing the frequency of one or more symptoms of a bleeding disorder or condition, such as a spontaneous or uncontrollable bleeding event. However, "treatment" does not need to be cured.

The term "perioperative management" as used herein means Long acting FIX polypeptides are used before, during or after surgical procedures such as surgery. Perioperative management The use of one or more bleeding events includes prior to surgery (ie, before surgery), during (ie, during surgery) or after (ie, after surgery) surgical prevention to prevent one or more bleeding or bleeding Events, or reduction or suppression of spontaneous and/or uncontrollable bleeding events before, during, and after surgery.

Pharmacokinetic (PK) parameters include the above terms and the following Language, unless otherwise indicated, has its ordinary meaning in the art. Some terms are explained in more detail in the examples. The PK parameter can be based on the FIX antigen content (usually interpreted as "antigen" in this article) Or the degree of FIX activity (usually referred to as "activity" in the form of a phrase in this article). PK parameters are often based on the extent of FIX activity in the literature due to the presence of endogenous inactive FIX in the plasma of some subjects that interferes with the ability to administer (ie, exogenous) FIX to antibodies using FIX. . However, when FIX is administered as part of a fusion protein or hybrid protein containing a heterologous polypeptide such as FcRn BP, the administered (i.e., exogenous) FIX antigen can be accurately measured using antibodies to the heterologous polypeptide. In addition, some PK parameters may be based on model prediction data (which is often referred to as "model prediction" in the form of interstitial sentences) or observational data (often interpreted as "observed" in this paper), and are preferably based on Observation data.

As used herein, "baseline" is the lowest measured plasma Factor IX content in a subject prior to administration of the dose. Factor IX plasma levels can be measured at two time points prior to dosing; at the time of screening visit and immediately prior to dosing. Alternatively, (a) a baseline for FIX activity <1% prior to treatment, a subject without detectable FIX antigen and a non-significant genotype may be defined as 0%, (b) a pre-treatment FIX activity <1% and The baseline of subjects who can detect FIX antigen can be set at 0.5%, and (c) the baseline of subjects with FIX activity between 1-2% before treatment is Cmin (the lowest activity throughout the PK study period), And (d) pre-treatment FIX activity The baseline of 2% of subjects can be set at 2%. Activity above the baseline prior to administration can be considered as a residual drug for prior treatment and can be attenuated to baseline after rFIXFc administration and subtracted from PK data.

As used herein, "T _1/2β " or "βHL" is the half-life associated with the elimination phase, t _1/2β = (ln2) / the elimination rate constant associated with the end stage. T _1/2β can be measured by FIX activity or the FIX antigen content in plasma. The activity-based T _{1/2β is} shown as T _1/2β (activity), and T _1/2β based on the FIX antigen content can be shown as T _1/2β (antigen). Both T _1/2β (activity) and T _1/2β (antigen) can be expressed as a range or geometric mean.

As used herein, "valley" is administered at a dose. The minimum level of plasma Factor IX activity achieved after the chimeric polypeptide of the invention or another Factor IX molecule and prior to administration of the next dose, if any. The bottom of the valley can be used interchangeably with the "limit" in this article. The baseline factor IX content was subtracted from the factor IX content to calculate the bottom content.

The term "annual bleeding rate"("ABR") as used herein refers to the number of bleeding events (including spontaneous and traumatic bleeding) experienced by a subject during a specified period of extrapolation to one year. For example, two bleedings within six months will indicate that the ABR is four. The median ABR provides a single value to describe all subjects, indicating that half of the subjects have individual ABRs less than or equal to the median and half of the ABRs are greater than or equal to the median. For example, the ABR can be calculated according to the following formula:

As used herein, "subject" means human. As this article The subject used includes a disease or condition known to have occurred at least one uncontrolled bleeding event, has been diagnosed with an uncontrolled bleeding event (eg, a bleeding disease or condition, such as hemophilia B), An individual with an uncontrolled bleeding event (such as hemophilia) or any combination thereof. Subject Individuals who are at risk of one or more uncontrolled bleeding events prior to an activity (eg, surgery, athletic activity, or any strenuous activity) may also be included. The subject's baseline FIX activity can be less than 1%, less than 0.5%, less than 2%, less than 2.5%, less than 3%, or less than 4%. Subjects also included human pediatrics. Pediatric human subjects are born to 20 years of age, preferably born to 18 years old, born to 16 years old, born to 15 years old, born to 12 years old, born to 11 years old, born to 6 years old, born to 5 years old Born to 2 years old and 2 to 11 years old.

As used herein, "therapeutic dose", "dose", "Effective dose" or "administration amount" means administration of a long-acting FIX polypeptide A dose of at least about 1 IU/dl or greater than 1 IU/dl of plasma FIX valer activity is achieved in the subject. For the purposes of the present invention, in one embodiment, "dose" means that the degree of plasma FIX valency activity is maintained at least about 1 IU/dl or greater than 1 IU/dl, at least about 2 IU/dl, during administration of the entire long acting FIX polypeptide. Or a dose greater than 2 IU/dl, at least about 3 IU/dl or greater than 3 IU/dl, at least about 4 IU/dl or greater than 4 IU/dl, or at least about 5 IU/dl or greater than 5 IU/dl. In another embodiment, the "dose" reduces or reduces the frequency of bleeding or bleeding disorders. In other embodiments, the "dose" stops an uncontrolled bleeding or bleeding event in progress. In other embodiments, the "dose" prevents such spontaneous bleeding or bleeding events in a subject susceptible to spontaneous bleeding or bleeding events. "Dose" or "therapeutic dose" does not require cure for hemophilia.

As used herein, "variant" means different from the original poly Nucleotide or polypeptide, but retains its essential properties (eg, factor IX clotting activity) Or a Fc (FcRn binding) activity) polynucleotide or polypeptide. In general, variants are generally closely similar and are identical to the original polynucleotide or polypeptide in many regions. Variants include polypeptides and polynucleotide fragments, deletions, insertions, and modifications of the original polypeptide.

II. Administration method

The invention provides a method of administering a long acting Factor IX (FIX) polypeptide to a human subject in need thereof, comprising administering to the subject a dose of a long acting FIX polypeptide at a certain dosing interval. Administration of a long acting FIX polypeptide is an alternative therapy achieved by the addition of recombinant FIX to a subject lacking FIX. Administration of a long-acting FIX polypeptide can reduce the number of bleeding or bleeding events in a subject or prevent a bleeding or bleeding event in a subject.

Subjects to which the methods of the invention are directed include those in need of control or prevention of bleeding or bleeding events. Subjects may be experiencing the following bleeding during administration, or are expected to experience the following bleeding, or may be susceptible to the following bleeding: a small amount of blood loss, joint blood, superficial muscle loss, soft tissue blood loss, moderate blood loss Intramuscular or soft tissue loss with anatomy, mucosal blood loss, hematuria, massive blood loss, pharyngeal blood loss, posterior pharyngeal blood loss, retroperitoneal blood loss, central nervous system blood loss, abrasion, incision, scratch, joint blood loss, nose bleeding, oral cavity Bleeding, bleeding gums, intracranial hemorrhage, intraperitoneal hemorrhage, a small amount of spontaneous blood loss, post-traumatic bleeding, moderate skin abrasions, or spontaneous blood loss to the joints, muscles, internal organs, or brain. Such subjects also include the need for perioperative management, such as managing bleeding associated with surgery or tooth extraction. In one embodiment, the subject needs to control one or A variety of bleeding events. In another embodiment, the subject requires individualized interval control. In other embodiments, the subject needs to treat one or more bleeding events as needed. In other embodiments, the subject requires a perioperative management of one or more bleeding events.

The invention also identifies that one or more bleeding disorders can be treated or prevented The appropriate dose and interval of administration. Administration of the amount of administration suitable for the dosing interval can achieve a plasma FIX trough activity level of at least about 1 IU/dl or greater than 1 IU/dl in the subject administered with the long acting FIX polypeptide during the interval. In one embodiment, the invention encompasses maintaining a plasma FIX trough activity level of at least about 1 IU/dl (1%) or greater than 1 IU/dl (1%), at least about 2 IU/dl (2%) throughout the interval. Or higher than 2 IU/dl (2%), at least about 3 IU/dl (3%) or higher than 3 IU/dl (3%), at least about 4 IU/dl (4%) or higher than 4 IU/dl (4%) Or, at least about 5 IU/dl (5%) or more than 5 IU/dl (5%), the amount of administration (or range of administration) and the interval of administration (or range of administration intervals). In another embodiment, the amount of administration (or range of administration) and the interval of administration (or range of administration intervals) reduce or decrease the frequency of bleeding or bleeding disorders. In other embodiments, the amount (or range of administration) of the long-acting FIX polypeptide and the interval of administration (or range of administration intervals) cause uncontrolled bleeding in the subject of administration of the administered amount. Or a bleeding event stops during the dosing interval. In other embodiments, the amount (or range of administration) of the long-acting FIX polypeptide and the interval of administration (or range of administration intervals) prevent such spontaneousness in a subject susceptible to spontaneous bleeding or bleeding events Bleeding or bleeding events. Various doses and dosing intervals are described in the international application filed on July 11, 2011 and published on January 12, 2012, as WO 2012/006624 In PCT/US2011/043569, the application is incorporated herein in its entirety by reference.

The dosage that can be used in the method of the invention is from about 10 IU/kg to About 200 IU/kg, from about 10 IU/kg to about 180 IU/kg, or from about 25 IU/kg to about 200 IU/kg. In one embodiment, the dosage of the long acting FIX polypeptide is from about 10 IU/kg to about 50 IU/kg, from about 10 IU/kg to about 100 IU/kg, from about 25 IU/kg to about 75 IU/kg, from about 25 IU/kg to about 100 IU. /kg, from about 25 IU/kg to about 125 IU/kg, from about 25 IU/kg to about 150 IU/kg, from about 25 IU/kg to about 50 IU/kg, from about 50 IU/kg to about 100 IU/kg, from about 50 IU/kg to about 150 IU /kg, from about 100 IU/kg to about 150 IU/kg, from about 150 IU/kg to about 200 IU/kg, or any combination thereof.

In another embodiment, at least about every five or five days or more The dosage of the long acting FIX polypeptide is as follows: from about 25 to about 110, from about 30 to about 110, from about 40 to about 110, from about 50 to about 110, from about 60 to about 110, from about 70 to about 110, from about 80 to about 110. , from about 90 to about 110, and from about 100 to about 110; from about 30 to about 100, from about 30 to about 90, from about 30 to about 80, from about 30 to about 70, from about 30 to about 60, from about 30 to about 50, From about 30 to about 40 IU/kg; from about 40 to about 110, from about 50 to about 100, from about 60 to about 90, and from about 70 to about 80 IU/kg; from about 40 to about 50, from about 50 to about 60, from about 60 to About 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, and about 100 to about 110 IU/kg; about 25, about 30, about 35, about 40, about 45, about 50, about 55 About 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105 or about 110 IU/kg.

In certain embodiments, the dose of the long acting FIX polypeptide is sufficient The amount of bleeding events that are controlled weekly. For example, a weekly dosing interval dose includes from about 10 to about 50, from about 20 to about 60, from about 20 to about 70, from about 20 to about 80, from about 20 to about 90, from about 30 to about 40, from about 30 to About 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 40 to about 50, about 40 to about 60, about 50 to about 70, about 40 to about 80 From about 40 to about 90, from about 50 to about 60, from about 50 to about 70, from about 50 to about 80, from about 50 to about 90, from about 60 to about 70, from about 60 to about 80, from about 60 to about 90, about 70 to about 80, about 70 to about 90 IU/kg. If effective for a given subject, the dosage can be less than 20 IU/kg, such as about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 IU/kg.

Alternatively, the dosing interval can be based on pharmacokinetic data or Additional information about the subject is determined by the individualized interval for each subject. The individualized dose/dosing interval combination may be the same as the dose/dosing interval combination of the fixed interval protocol in the previous paragraph, or may be different. The protocol can be initially at a fixed dosing interval, which can then be changed to an individualized dosing interval.

In some embodiments, the dose of the long acting FIX polypeptide is sufficient The amount of bleeding events was controlled at individualized intervals. In one example, the individualization interval is every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19th or twice a month. Examples of dosages for individualized intervals include, but are not limited to, from about 50 IU/kg to about 180 IU/kg, from about 60 IU/kg to about 180 IU/kg, from about 70 IU/kg to about 180 IU/kg, from about 80 IU/kg to about 180 IU/kg, from about 90 IU/kg to about 180 IU/kg, from about 100 IU/kg to about 180 IU/kg, from about 110 IU/kg to about 180 IU/kg, from about 120 IU/kg to about 180 IU/kg, from about 130 IU/kg to about 180 IU/kg, from about 140 IU/kg to about 180 IU/kg, from about 60 IU/kg to about 170 IU/kg, from about 60 IU/kg to about 160 IU/kg, from about 60 IU/kg to about 150 IU/kg, from about 60 IU/kg to about 140 IU/kg, from about 70 IU/kg to about 140 IU/kg, from about 70 IU/kg to about 130 IU/kg, from about 80 IU/kg to about 130 IU/kg, from about 80 IU/kg to about 140 IU/kg, from about 90 IU/kg to about 140 IU/kg, from about 90 IU/kg to about 130 IU/kg, from about 90 IU/kg to about 120 IU/kg, from about 90 IU/kg to about 110 IU/kg, from about 90 IU/kg to about 100 IU/kg, from about 100 IU/kg to about 140 IU/kg, from about 100 IU/kg to about 130 IU/kg, from about 100 IU/kg to about 120 IU/kg, or from about 100 IU/kg to about 110 IU/kg.

In other embodiments, the individualized dosing interval is at least about Within two weeks, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about three weeks, at least about 22 Day, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, or at least about one month. For example, the dosage of the individualized dosing interval is from about 120 IU/kg to about 200 IU/kg, from about 130 IU/kg to about 200 IU/kg, from about 140 IU/kg to about 200 IU/kg from about 150 IU/kg to about 200 IU/kg. From about 160 IU/kg to about 200 IU/kg, from about 170 IU/kg to about 200 IU/kg, from about 180 IU/kg to about 200 IU/kg, from about 120 IU/kg to About 180 IU/kg, from about 130 IU/kg to about 180 IU/kg, from about 140 IU/kg to about 180 IU/kg, from about 150 IU/kg to about 180 IU/kg, from about 160 IU/kg to about 180 IU/kg, from about 120 IU/kg to About 170 IU/kg, about 130 IU/kg to about 170 IU/kg, about 140 IU/kg to about 170 IU/kg, about 150 IU/kg to about 170 IU/kg, about 160 IU/kg to about 170 IU/kg, about 120 IU/kg to About 160 IU/kg, from about 130 IU/kg to about 160 IU/kg, from about 140 IU/kg to about 160 IU/kg, from about 150 IU/kg to about 160 IU/kg, from about 120 IU/kg to about 150 IU/kg, from about 130 IU/kg to About 150 IU/kg, from about 140 IU/kg to about 150 IU/kg, from about 120 IU/kg to about 140 IU/kg, or from about 130 IU/kg to about 140 IU/kg. In other examples, the dosage of the individualized dosing interval is about 60 IU/kg, about 70 IU/kg, about 80 IU/kg, about 90 IU/kg, about 95 IU/kg, about 100 IU/kg, about 105 IU/kg, about 110 IU. /kg, about 115 IU/kg, about 120 IU/kg, about 125 IU/kg, about 130 IU/kg, about 135 IU/kg, about 140 IU/kg, about 145 IU/kg, about 150 IU/kg, about 155 IU/kg, about 160 IU. /kg, about 165 IU/kg, about 170 IU/kg, about 175 IU/kg or about 180 IU/kg.

In some embodiments, the dose of the long acting FIX polypeptide is sufficient Treat one or more bleeding events as needed. The dose to be treated on demand may vary depending on various factors such as the subject's baseline FIX activity, the weight of the subject, the likelihood of the subject experiencing a bleeding event, and the like. In one example, the dosage of the on-demand treatment can be from about 10 to about 50, from about 15 to about 100, from about 20 to about 100, from about 20 to about 50, from about 50 to about 100, from about 10, from about 20, from about 40. , about 50 and about 100 IU/kg. In another example, an on-demand treatment agent The amount may be from about 20 to about 50, from about 20 to about 100, from about 20 to about 180, from 25 to about 110, from about 30 to about 110, from about 40 to about 110, from about 50 to about 110, from about 60 to about 110, From about 70 to about 110, from about 80 to about 110, from about 90 to about 110, from about 100 to about 110, from about 30 to about 100, from about 30 to about 90, from about 30 to about 80, from about 30 to about 70, from about 30 Up to about 60, about 30 to about 50, about 30 to about 40 IU/kg, about 40 to about 110, about 50 to about 100, about 60 to about 90, about 70 to about 80 IU/kg, about 40 to about 50, From about 50 to about 60, from about 60 to about 70, from about 70 to about 80, from about 80 to about 90, from about 90 to about 100, from about 100 to about 110 IU/kg, from about 20, from about 25, from about 30, from about 35, About 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, and about 110 IU/kg. In other examples, the dosage of on-demand treatment can range from about 90 to about 180, from about 100 to about 180, from about 110 to about 180, from about 120 to about 180, from about 130 to about 180, from about 140 to about 180, from about 150. To about 180, about 160 to about 180, and about 170 to about 180 IU/kg. In other examples, the dosage of the on-demand treatment is from about 90 to about 170, from about 90 to about 160, from about 90 to about 150, from about 90 to about 140, from about 90 to about 130, from about 90 to about 120, from about 90 to About 110, and about 90 to about 100 IU/kg. In an example, the dosage of the on-demand treatment is from about 100 to about 170, from about 110 to about 160, from about 120 to about 150, and from about 130 to about 140 IU/kg. In other examples, the dosage of on-demand treatment is from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 130 to about 140, from about 140 to about 150, from about 150 to About 160, and about 160 to about 170 IU/kg. The dose of on-demand treatment can be about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, and about 180 IU/kg.

In certain embodiments, the amount administered to the subject is given The drug compartment combination was 20 IU/kg once a week, 40 IU/kg once a week, 50 IU/kg once a week, 100 IU/kg every 10 days, and 100 IU/kg every two weeks (or twice a month). Other combinations of dose and dose interval include: a dose of at least about 50 IU/kg and a dosing interval of at least about 7 days, a dose of at least about 100 IU/kg, a dosing interval of at least about 9 days, a dose of at least about 100 IU/kg, and a dosing interval of at least Approximately 12 days, a dose of at least about 100 IU/kg and a dosing interval of at least about 13 days, a dose of at least about 100 IU/kg, a dosing interval of at least about 14 days, a dose of at least about 100 IU/kg, and a dosing interval of at least about 15 days, at a dose At least about 150 IU/kg and a dosing interval of at least about 14 days, 20-50 or 20-100 IU/kg and the dosing interval is once a week, the dose is 20-50 IU/kg, and the dosing interval is 7 days, the dose is 50-100 IU. /kg and dosing interval 10-14 days, or dose 100-150 IU / kg and dosing interval 14-16 days. Non-limiting examples of combinations of dosing intervals and dosages also include 10-50 IU/kg 7 days, 15-100 IU/kg 10-13 days, 50-150 IU/kg 14-15 days, 10-30 IU/kg 7 days, 15-50 IU/kg 10 days, 20-70 IU/kg 11 days, 25-85 IU/kg 12 days, 30 to 100 IU/kg 13 days, 40 to 125 IU/kg 14 days, and 50-150 IU/kg 15 days.

In one embodiment, the dosing interval is at least about one week Second, at least about two weeks, at least about one month, at least about three weeks, at least about four weeks, or at least about once a month. In another In embodiments, the dosing interval of the long acting FIX polypeptide is from about once every five days to about once every two months, once within about five days to once within about one month, or from about once a week to about once a month. In other embodiments, the dosing interval is at least about every 5 days, about every 6 days, at least about every 7 days, at least about every 8 days, at least about every 9 days, at least about every 10 days, at least about every 11 days. At least about every 12 days, at least about every 13 days, at least about every 14 days, at least about every 15 days, at least about every 16 days, at least about every 17 days, at least about every 18 days, at least about every 19 days, at least About every 20 days or at least about every 21 days. In other embodiments, the dosing interval is 9-18 days, such as about 9-17, about 9-16, about 9-15, about 9-14, about 9-13, about 9-12, about 9-11 , about 9-10 days, about 10-18, about 11-18, about 12-18, about 13-18, about 14-18, about 15-18, about 16-18, about 17-18 days, about 10 -11, about 11-12, about 12-13, about 13-14, about 14-15, about 15-16, and about 16-17 days, about 9, about 10, about 11, about 12, about 13, About 14, about 15, about 16, about 17, or about 18 days. In other embodiments, the dosing interval is about 10-14 days. The dosing interval may be longer than 18 days, such as about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32. , about 33, about 34, about 35, about 36, about 37, about 38, about 39 or about 40 days.

In one embodiment, the frequency of administration of the long acting FIX polypeptide It is about every two weeks, or twice a month. In another embodiment, the frequency of administration is 25-50 IU/kg every 7 days, 50-100 IU/kg every 10-13 days, or 100-150 IU/kg every 14 days. Determining the interval (or frequency) and dose such that the combination of interval (or frequency) and dose will produce at least about 1-5 or at least in the subject. A degree of FIX valency of from about 1-3 or at least about 1, at least about 2, at least about 3 IU/dl. Examples of the dose and administration interval may also be 20-50 IU/kg on the 7th, 50-100 IU/kg on the 10-14th, 100-150 IU/kg on the 14-16th, 10-50 IU/kg on the 7th, and 10-13 days. 15-100 IU/kg, or 14-15 days 50-150 IU/kg. Other examples of dosages and dosing intervals include, but are not limited to, 10-30 IU/kg on the 7th, 15-50 IU/kg on the 10th, 20-70 IU/kg on the 11th, 25-85 IU/kg on the 12th, and 30 on the 13th. 100 IU/kg, 40 to 125 IU/kg on the 14th, and 50-150 IU/kg on the 15th.

In some embodiments, the dosing interval is once a week 20 IU/kg, 40 IU/kg every 10 days, 50 IU/kg once a week or 100 IU/kg every two weeks (twice a month).

In some embodiments of the invention, the year of the bleeding event The bleeding rate (ABR) is controlled by the method of the invention. For example, the amount administered and the interval of administration can be administered to reduce or reduce the annual bleeding rate to some extent. In one embodiment, the administration of the long-acting FIX polypeptide at a dose and administration interval for controlling a bleeding event results in an annual bleeding rate of less than 2, less than 2.5, less than 3, less than 3.5, less than 4, less than 4.5, less than 5, Less than 5.5, less than 6, less than 6.5, less than 7, less than 7.5, less than 8, less than 8.5, less than 9, less than 9.5 or less than 10. For example, the weekly ABR for controlling bleeding events can be 2.95. In another embodiment, the administration of the long acting FIX polypeptide at a dose and dosing interval for individualized interval control bleeding events such that the ABR is less than 1, less than 1.5, less than 2, less than 2.5, less than 3, less than 3.5, less than 4. Less than 4.5, less than 5, less than 5.5, less than 6, less than 6.5, less than 7, less than 7.5, less than 8, less than 8.5 or less than 9. For example, the individualized interval control ABR can be 1.38. In other embodiments, the administration of the long acting FIX polypeptide at a dose and dosing interval for an on-demand (ie, paroxysmal) bleeding event results in an ABR of less than 10, less than 11, less than 12, less than 13, less than 14. Less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, or less than 26. For example, the on-demand ABR can be 17.69.

Long FIX polypeptides of the present invention may provide longer than wild type FIX (i.e. by SEQ ID NO: 2 polypeptide consisting of amino acids 1-415 of; BENEFIX ^®; or pdFIX) of the half-life, for example, _{T 1 /} 2β (active) Or T _1/2β (antigen). In one embodiment, the long-acting FIX polypeptide has a T _{1/2 β} (activity) of at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least About 70 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, at least about 100 hours, at least about 105 hours, at least about 110 hours, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, At least about 185, at least about 190, or at least about 193. In another embodiment, the long-acting FIX polypeptide has a T _{1/2 β} (activity) of from about 40 hours to about 193 hours, from about 35 hours to about 190 hours, from about 45 hours to about 193 hours, from about 50 hours to about 198. Hours, from about 55 hours to about 188 hours, from about 60 hours to about 200 hours, from about 30 hours to about 205 hours, from about 43 hours to about 210 hours. In a particular embodiment, T _{1/2 β} (activity) is from about 40 hours to about 193 hours.

In some embodiments, the T _{1/2 β} (activity) of the long-acting FIX polypeptide is expressed as an average. For example, the average of T _{1/2 β} (activity) of the long-acting FIX polypeptide is at least about 76 hours, at least about 77 hours, at least about 78 hours, at least about 79 hours, at least about 80 hours, at least about 81 hours, At least about 82 hours, at least about 83 hours, at least about 84 hours, at least about 85 hours, at least about 86 hours, at least about 87 hours, at least about 88 hours, at least about 89 hours, at least about 90 hours, at least about 91 hours, or At least about 92 hours. In a specific embodiment, the average of T _{1/2 β} (activity) of the long-acting FIX polypeptide is 82 hours.

In other embodiments, the FIX polypeptides of long-term T _{1 / 2β} (active) line in the wild type mature FIX (i.e. BENEFIX ^®) is done by comparing the sum T _{1 / 2β} (activity) to be displayed. In one _example, T _{1 /} 2β (activity) of the wild-type mature FIX average ratio (by the SEQ ID NO: 2 amino acid composition of the 1-415 polypeptide, i.e. BENEFIX ^®; or pdFIX) is at least about 2.0 Times. In another _example, T _{1 /} 2β (activity) of the wild-type mature FIX average ratio (by the SEQ ID NO: 2 amino acids 1-415 of the polypeptide composition, i.e. BENEFIX ^®; pdFIX) at least about 2.0倍, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0 Times, at least about 3.1 times or at least about 3.2 times.

In other embodiments, the invention provides for administration of a long acting FIX polypeptide to a subject that exhibits a longer T _{1/2 β} (activity). In one embodiment, the method of the invention comprises exhibiting T _{1/2 β} (activity) for at least about 80 hours, at least about 81 hours, at least about 82 hours, at least about 83 hours, at least about 84 hours, at least about 85 hours, Subjects that are at least about 86 hours, at least about 87 hours, at least about 88 hours are administered a long acting FIX polypeptide. In another embodiment, the method comprises administering to the subject having a baseline FIX activity of less than 2%, less than 1.5%, less than 1%, or less than 0.5% a long-acting FIX polypeptide, thereby resulting in a high T _{1/2 β} (activity) About 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, or about 88 hours.

In certain embodiments, the long-acting FIX polypeptide has a T _{1/2 β} (antigen) of at least about 63 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, At least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, at least about 190 hours, at least about 200 hours, at least about 210, at least About 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340 At least about 350, at least about 360, or at least about 370. In another embodiment, the T _{1/2 β} (antigen) of the long-acting FIX polypeptide is from about 63 hours to about 372 hours, from about 50 hours to about 380 hours, from about 40 hours to about 390 hours, from about 70 hours to about 360. Hours, from about 80 hours to about 400 hours, from about 90 hours to about 410 hours, from about 50 hours to about 400 hours, from about 40 hours to about 380 hours. In a particular embodiment, the T _{1/2 β} (antigen) is from about 63 hours to about 372 hours.

In some embodiments, the T _{1/2 β} (antigen) of the long-acting FIX polypeptide is represented as an average. For example, the average of the T _{1/2 β} (antigen) of the long-acting FIX polypeptide is at least about 110 hours, at least about 111 hours, at least about 112 hours, at least about 113 hours, at least about 114 hours, at least about 115 hours, At least about 116 hours, at least about 117 hours, at least about 118 hours, at least about 119 hours, at least about 120 hours, at least about 121 hours, at least about 122 hours, at least about 123 hours, at least about 124 hours, at least about 125 hours, At least about 126 hours, at least about 127 hours, at least about 128 hours, at least about 129 hours, at least about 130 hours, or at least about 131 hours. In a particular embodiment, the average of T _{1/2 β} (antigen) of the long-acting FIX polypeptide is 118 hours or 126 hours.

In other embodiments, the FIX polypeptides of long-term _{T 1 /} 2β (antigen) system in the wild type mature FIX (of SEQ ID NO: 2 polypeptide consisting of amino acids 1-415 of, i.e. BENEFIX ^® or pdFIX) The T _1/2β (antigen) is displayed in a comparison manner. In one _example, T _{1 /} 2β (antigen) than the average wild-type mature FIX (the SEQ ID NO: 2 amino acids 1-415 of the polypeptide composition, i.e. BENEFIX ^®; or pdFIX) is at least about 2.0 Times. In another _example, T _{1 /} 2β (antigen) than the average wild-type mature FIX (the SEQ ID NO: 2 amino acid composition of the 1-415 polypeptide, i.e. BENEFIX ^®; pdFIX) at least about 2.0倍, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 Times.

In other embodiments, the invention provides for administration of a long acting FIX polypeptide to a subject that exhibits a longer T _{1/2 β} (antigen). In one embodiment, the method of the invention comprises exhibiting T _{1/2 β} (antigen) for at least about 115 hours, at least about 116 hours, at least about 117 hours, at least about 118 hours, at least about 119 hours, at least about 120 hours, The subject is administered a long-acting FIX polypeptide for at least about 121 hours, at least about 122 hours, at least about 123 hours, at least about 124 hours, at least about 125 hours, at least about 126 hours, or at least about 127 hours. In another embodiment, the method comprises administering to the subject having a baseline FIX activity of less than 2%, less than 1.5%, less than 1%, or less than 0.5% a long-acting FIX polypeptide, thereby resulting in a high T _{1/2 β} (activity) About 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, about 120 hours, about 121 hours, about 122 hours, about 123 hours, about 124 hours, about 125 hours, about 126 hours, about 127 hours, about 128 hours or about 129 hours.

In certain embodiments, T _{1/2 β} (activity) is measured by single-stage coagulation analysis.

In other embodiments, the long-acting FIX polypeptide has an average residence time (MRT) of at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, or at least about 190 hours. In some embodiments, the MRT is from about 50 hours to about 200 hours, from about 60 hours to about 210 hours, from about 60 hours to about 183 hours, from about 70 hours to about 150 hours, from about 70 hours to about 140 hours, about 70 Hours to about 130 hours, from about 80 hours to about 120 hours, from about 80 hours to about 110 hours, or from about 88 hours to about 110 hours. In a particular embodiment, the ratio of the MRT SEQ ID NO: 2 amino acids 1-415 of the polypeptide or composition of the high BENEFIX ^® at least about 2.0 fold (e.g., 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.7 times, 2.8 times, 2.9 times or 3 times).

In some embodiments of the invention, the method of the invention proceeds to The step comprises measuring the subject's baseline FIX activity prior to initial administration of the long acting FIX polypeptide. Measurement of baseline FIX activity can be performed by any known coagulation assay in the art, such as single-step aPTT analysis, two-step color analysis, ROTEM, TGA, and the like.

In some embodiments, the methods of the invention further comprise measuring T _{1/2 β} (activity) or T _{1/2 β} (antigen) of the subject's long-acting FIX polypeptide after administration of the long-acting FIX polypeptide.

In other embodiments, one or more established doses are administered After the long-acting FIX polypeptide, the plasma trough content of the subject's long-acting FIX polypeptide is maintained at about 1 IU/dl (1%) or greater than 1 IU/dl (1%). In other embodiments, the plasma trough content of the subject's long acting FIX polypeptide is maintained between about 1 IU/dl (1%) and about 5 IU/dl (5%). In other embodiments, the plasma trough content is maintained between about 1% and about 5%, between about 1% and about 6%, between about 1% and about 7%, at about 1% and about 8 Between %, between about 1% and about 9%, between about 1% and about 10%, at about 1% and about Between 12%, between about 1% and about 14%, between about 1% and about 15%, between about 1% and about 17%, between about 1% and about 19%, at Between about 1% and about 20%, between about 1% and about 22%, between about 1% and about 24%, between about 1% and about 25%, at about 1% and about 30% Between %, between about 1% and about 35%.

In some embodiments, after administration of a long acting FIX polypeptide After about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days, the bottom is 1-5 IU/dl or 1-3 IU/dl. In some embodiments, the plasma content of the subject's FIX polypeptide reaches an average trough of at least about 1 IU/dl after at least about 6 days, or at least about 1, 2, 3, 4, or 5 IU after at least about 6 days. / dl bottom. In some embodiments, the chimeric polypeptide has a plasma content of up to about 1-5 IU/dl or an average trough of 1-3 IU/dl. The bottom or average valley may be at about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, It is reached after about 37, about 38, about 39 or about 40 days.

III. Long-acting FIX polypeptide

Long acting or long lasting FIX polypeptides suitable for use in the present invention are chimeric polypeptides comprising a FIX polypeptide and an FcRn binding partner. In certain embodiments, rFIXFc is a single human recombinant coagulation FIX (rFIX) comprising a dimeric Fc domain covalently linked to immunoglobulin G1 (IgG1) in a non-intervening sequence Molecular fusion protein. Unless otherwise specified, a FIX polypeptide of the invention comprises a functional Factor IX polypeptide that exerts its normal clotting action. Thus, FIX polypeptides include functional variant polypeptides and polynucleotides encoding such functional variant polypeptides. In one embodiment, the FIX polypeptide is a human, bovine, porcine, canine, feline, and murine FIX polypeptide. A full range of polypeptides and polynucleotide sequences of FIX are known, and a number of functional variants, such as fragments, mutants and modified forms, are also known. The FIX polypeptide includes full length FIX, minus full length FIX of Met at the N-terminus, full length FIX minus the signal sequence, mature FIX (minus signal sequence and propeptide), and maturation at the N-terminus with another Met FIX. FIX can be prepared by recombinant means ("Recombinant Factor IX" or "rFIX"), ie it is not naturally occurring or derived from plasma.

Many functional FIX variants are known. Quoted in full text The International Publication No. WO 02/040544 A3, which is incorporated herein by reference, discloses the mutants which exhibit an increased resistance to inhibition by heparin, on page 4, columns 9-30, and page 15, columns 6-31. International Publication No. WO 03/020764 A2, which is incorporated herein by reference in its entirety, discloses T-cell immunogens in Tables 2 and 3 (on pages 14-24) and on page 12, columns 1-27. Reduced FIX mutants. International Publication No. WO 2007/149406 A2, which is hereby incorporated by reference in its entirety, discloses the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of A functional mutant FIX molecule with increased resistance to proteases. WO 2007/149406 A2 also discloses chimeric and other variant FIX molecules on page 19, column 12 to page 20, column 9. International Publication No. WO 08/118507 A2, which is incorporated herein by reference in its entirety, is incorporated herein by reference. Columns 14 through 6 of column 5 reveal FIX mutants exhibiting increased coagulation activity. International Publication No. WO 09/051717 A2, which is hereby incorporated by reference in its entirety, in the entire disclosure of the disclosure of the disclosure of the disclosure of FIX mutants linked and/or O-linked to a glycosylation site. International Publication No. WO 09/137254 A2, which is incorporated herein by reference in its entirety, also on page 2, paragraphs [006] to 5, paragraph [011], and page 16, paragraph [044] to Paragraph [057] on page 24 discloses a Factor IX mutant with increased number of glycosylation sites. International Publication No. WO 09/130198 A2, which is hereby incorporated by reference in its entirety, in the entire disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of Mutant FIX molecule. International Publication No. WO 09/140015 A2, which is incorporated herein by reference in its entirety, is hereby incorporated by reference in the paragraphs [0043] to 13 [0053] on page 11 for use in the conjugation of polymers (e.g., PEG). A functional FIX mutant with an increased number of Cys residues. The FIX polypeptides described in International Application No. PCT/US2011/043569, filed on Jan. 11, 2011, which is hereby incorporated by reference in its entirety, in

In addition, hundreds of FIX have been identified in hemophilia subjects. Non-functional mutations, many of which are disclosed in Table 5 on pages 11-14 of International Publication No. WO 09/137254 A2, which is incorporated herein by reference in its entirety. Such non-functional mutations are not included in the present invention, but provide additional guidance as to which mutations are more likely or less likely to produce a functional FIX polypeptide.

In one embodiment, Factor IX (or the cause of the chimeric polypeptide) Sub-IX part) FIX amino acid sequence (amino acid 1 to 415 of SEQ ID NO: 2) which has no signal sequence and propeptide sequence shown in Table 18A, or FIX amino acid having a propeptide sequence a sequence, or a FIX amino acid sequence (full length FIX) having a propeptide and a signal sequence of at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% At least 99% or 100% consistent.

Factor IX coagulation activity is expressed in International Units (IU). The FIX activity of one IU approximately corresponds to the amount of FIX in one milliliter of normal human plasma. Several assays can be used to measure factor IX activity, including single-stage coagulation analysis (activated partial clotting hormone time; aPTT), thrombin generation time (TGA), and rotational thrombosis (ROTEM ^® ). See, for example, Example 3.

Unless otherwise specified, the FcRn binding partner ("FcRn BP") contains a functional neonatal Fc receptor (FcRn) binding partner. The FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor and subsequently actively transported by the FcRn receptor of the FcRn binding partner. Thus, the term FcRn BP includes any functional variant of IgG Fc. For example, the region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372: 379, which is incorporated herein by reference in its entirety). The main contact region of Fc and FcRn is close to the junction of the CH2 domain and the CH3 domain. The Fc-FcRn contacts are all within a single Ig heavy chain. FcRn BP includes intact IgG, Fc fragments of IgG, and other fragments of IgG including the entire binding region of FcRn. The main contact sites include amino acid residues 248, 250-257 of the CH2 domain, Amino acid residues 385-387, 428 and 433-436 of 272, 285, 288, 290-291, 308-311 and 314, and the CH3 domain. References to amino acid numbers of immunoglobulin or immunoglobulin fragments or regions are based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda; MD, which is incorporated by reference in its entirety. Incorporated herein. (FcRn receptors have been isolated from several mammalian species including humans. Sequences of human FcRn, rat FcRn and mouse FcRn are known (Story et al., 1994, J. Exp. Med. 180: 2377), The manner of full reference is incorporated herein). FcRn BP may comprise the CH2 and CH3 domains of the immunoglobulin with or without the hinge region of the immunoglobulin. Exemplary FcRn BP variants are provided in WO 2004/101740 and WO 2006/074199, which are incorporated herein by reference in their entirety.

FcRn BP (or the FcRn BP portion of the chimeric polypeptide) may contain One or more mutations and combinations of mutations.

FcRn BP (or the FcRn BP portion of the chimeric polypeptide) may contain Mutations that confer increased half-life, such as M252Y, S254T, T256E, and combinations thereof, as disclosed in Oganesyan et al., Mol. Immunol. 46: 1750 (2009), incorporated herein by reference in its entirety; H433K, N434F and Combinations, as disclosed in Vaccaro et al., Nat. Biotechnol. 23: 1283 (2005), which is incorporated herein by reference in its entirety herein in its entirety in its entirety in - page 2 [0012] and the mutants disclosed in Examples 9 and 10; and US20090163699 A1, which is incorporated herein by reference in its entirety. The mutants disclosed in paragraphs [0014] to [0021] on page 2.

FcRn BP (or the FcRn BP portion of the chimeric polypeptide) may also be included The following mutations are included: the Fc region of IgG can be modified according to well-accepted procedures such as site-directed mutagenesis and similar procedures to produce a modified IgG or Fc fragment or portion thereof to be bound by FcRn. Such modifications include modifications that retain or even enhance binding to FcRn away from the FcRn contact site and modifications within the contact site. For example, the following single amino acid residues in human IgG1 Fc (Fcy1) can be substituted without significantly losing the binding affinity of Fc for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A and K447A, wherein, for example, P238A represents wild type proline acid substituted with alanine at position number 238. In addition to alanine, other amino acids may also be substituted for the wild type amino acid at the above specified positions. Mutations can be introduced into the Fc one by one, resulting in more than one hundred FcRn binding partners different from the native Fc. Alternatively, a combination of two, three or more of these individual mutations can be introduced together to generate hundreds of additional FcRn binding partners. Some of these mutations confer new functions on the FcRn binding partner. For example, one embodiment has N297A to remove highly conserved N-glycosylation sites. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the FcRn binding partner and rendering the FcRn binding partner unable to bind to FcyRI, FcyRIIA, FcyRIIB and FcyRIIIA without impairing affinity for FcRn (Routledge et al., 1995). Transplantation 60: 847, which is incorporated herein by reference in its entirety; Friend et al., 1999, Transplantation 68: 1632, which is incorporated herein by reference in its entirety; Shields et al., 1995, J. Biol. .276:6591, which is incorporated herein by reference in its entirety. In addition, at least three human Fc gamma receptors appear to recognize the binding site on the IgG in the lower hinge region, typically the amino acid 234-237. Thus, another example of a novel function and potentially reduced immunogenicity can be produced by mutations in this region, for example by replacing the amino acid 233-236 "ELLG" of human IgG1 with the corresponding sequence "PVA" from IgG2. (There is a loss of amino acid) to produce. It has been shown that when these mutations have been introduced, FcyRI, FcyRII and FcyRIII, which mediate various effector functions, will not bind. In IgG1 (Ward and Ghetie, 1995, Therapeutic Immunology 2:77, which is incorporated herein by reference in its entirety; and by Armour et al., 1999, Eur. J. Immunol. 29:2613, which is incorporated by reference in its entirety Into this article). As another example of the novel function resulting from the above mutations, the affinity for FcRn can be increased to exceed the affinity of the wild type in some cases. This increase in affinity may reflect an increase in the "adsorption" rate, a decrease in the "desorption" rate, or an increase in the "adsorption" rate and a decrease in the "desorption" rate. Mutations conferring increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al, 2001, J. Biol. Chem. 276:6591, which is incorporated herein by reference in its entirety).

FcRn BP (or FcRn BP portion of a chimeric polypeptide) can be linked to a table The Fc amino acid sequence of the signalless sequence shown in 18A or Table 18B (amino acid 1 to 227 of SEQ ID NO: 2) or the Fc amino acid sequence having the signal sequence is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

Chimeric polypeptide comprising a FIX polypeptide and an FcRn binding partner Can comprise Fc amino acid sequence (SEQ ID NO: 2 amino acid 1 to 642) with Factor IX and FcRn BP (for example, the signalless and propeptide sequences shown in Table 18A, or having a propeptide sequence) The Fc amino acid sequence, or the Fc amino acid sequence having the signal sequence and the propeptide sequence) is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence Column.

A long acting or long lasting FIX polypeptide can be a hybrid FIX polypeptide. miscellaneous Crossed FIX polypeptide means a combination of a FIX chimeric polypeptide and a second polypeptide. The chimeric polypeptide in the hybrid can be associated with the second polypeptide via non-covalent protein-protein interactions, such as charge-charge or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in the hybrid can be associated with each other via a covalent bond such as a disulfide bond. The chimeric peptide and the second peptide can be associated with each other via one or more types of bonds, such as non-covalent bonds and disulfide bonds. The hybrids are described in WO 2004/101740, WO 2005/001025, U.S. Patent No. 7,404,956, U.S. Patent No. 7,348,004, and WO 2006/074, each incorporated herein by reference. The second polypeptide can be a second copy of the same chimeric polypeptide or it can be a different chimeric polypeptide. In other embodiments, the second polypeptide is a polypeptide comprising an FcRn BP such as Fc. In some embodiments, the chimeric polypeptide is Factor IX-FcRn BP, eg, a Factor IX-Fc chimeric polypeptide, and the second polypeptide consists essentially of Fc. See, for example, Table 18 (SEQ ID NOS: 2 and 4). See, for example, US 7,404,956, incorporated herein by reference in its entirety.

The second polypeptide in the hybrid may comprise or consist essentially of The following composition: amino acid sequence with no signal sequence shown in Table 18B (amino acid 1 to 227 of SEQ ID NO: 4) or amino acid sequence having a signal sequence of at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence.

In some embodiments, the long acting FIX polypeptide is a FIX monomer Dimer hybrids. The monomer-dimer hybrid may comprise two polypeptide chains, one chain comprising a FIX polypeptide and a first Fc region, and the other chain comprising a second Fc region, consisting essentially of a second Fc region or by a second Fc region composition. In certain aspects, the FIX monomeric dimer hybrid consists essentially of or consists of two polypeptide chains, the first strand consisting essentially of or consisting of a FIX polypeptide, and the second strand Substantially consists of or consists of a second Fc region.

Long acting FIX polypeptides can be with, for example, SEQ ID NO: 1 or 3 a nucleotide coding sequence (individual or a combination of a Factor IX portion, an Fc portion) or a complementary strand thereof, a known mutation, and a nucleotide coding sequence of a recombinant Factor IX or Fc (such as disclosed in the publications and patents cited herein) a revealer, or a complementary strand thereof, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4 (individual or a combination of a Factor IX portion, an Fc portion) and/or a polynucleotide fragment of any such nucleic acid molecule ( For example, the fragments described herein are at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequences encoded. Also included as a variant, a polynucleotide which hybridizes to such nucleic acid molecules under strict hybridization conditions or a lower stringency condition, and also a polypeptide encoded by such a polynucleotide, as long as it has functionality.

A nucleotide sequence having a nucleic acid having at least 95% "consistent" with a reference nucleotide sequence is intended to mean that the nucleotide sequence of the nucleic acid is identical to the reference sequence, with the exception that every 100 nucleotides of the reference nucleotide sequence The nucleotide sequence can include up to five point mutations. In other words, to obtain a nucleic acid whose nucleotide sequence is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or taken from another nucleotide. Up to 5% of the nucleotides of the total nucleotides in the generation or reference sequence can be inserted into the reference sequence. The query sequence can be, for example, the entire sequence shown in SEQ ID NO: 1 or 3, the ORF (open reading frame) or any of the specified fragments as described herein.

In fact, whether any particular nucleic acid molecule or polypeptide is associated with this The nucleotide sequence or polypeptide of the invention is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical and can be determined routinely using known computer programs. A preferred method for determining the best overall match between a query sequence (reference sequence or original sequence) and the target sequence (also known as overall sequence alignment) can be used based on Brutlag et al. (Comp. App. Biosci. 1990) 6: 237-245) The FASTDB computer program is used to determine the algorithm, which is incorporated herein by reference in its entirety. In sequence alignment, both the query sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The results of the overall sequence alignment are expressed as percent identity. The preferred parameters for calculating the percent identity in the FASTDB DNA sequence alignment are: matrix = single matrix, k-tuple = 4, mismatch penalty = 1, junction penalty = 30, randomization group length = 0 , truncation score = 1, gap penalty = 5, gap size penalty of 0.05, window size = 500 or the length of the target nucleotide sequence, whichever is shorter.

If the target sequence is missing due to 5' or 3' rather than due to internal defects If the error is shorter than the query sequence, the result must be manually corrected. This is because the FASTDB program does not consider the 5' and 3' truncation of the target sequence when calculating the percent consistency. For a sequence that is truncated at the 5' or 3' end relative to the query sequence, the query is calculated as a percentage of the total base of the query sequence The sequence is corrected for the percent identity by the number of unmatched/aligned bases 5' and 3' of the target sequence. Whether the nucleotides match/align are determined by the FASTDB sequence alignment results. This percentage is then subtracted from the percentage of consistency calculated using the FASTDB program above the specified parameters to obtain a final consistency percentage score. This corrected score is divided into scores for the purpose of the present invention. For the purpose of manually adjusting the percent consistency score, only bases outside the 5' and 3' bases of the target sequence that are not matched/aligned with the query sequence are calculated, as indicated by the FASTDB alignment.

For example, a sequence of 90 bases and 100 sequences The base query sequence is aligned to determine the percent identity. The deletion is present at the 5' end of the target sequence, so the FASTDB alignment does not show the match/alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of unmatched bases at the 5' and 3' ends / total number of bases in the query sequence), thus subtracted from the percent identity score calculated by the FASTDB program 10%. If the remaining 90 bases match exactly, the final percent identity will be 90%. In another example, a sequence of 90 bases is compared to a 100 base query sequence. This time, the deletion is an internal deletion, so there are no bases on the 5' or 3' of the target sequence that are not matched/aligned with the query sequence. In this case, the percent consistency calculated by FASTDB is not manually corrected. Again, only the 5' and 3' bases of the target sequence that are not matched/aligned with the query sequence are manually corrected. For the purposes of the present invention, no other manual corrections will be made.

The polypeptide has at least one example of the amino acid sequence of the present invention Amino acid sequence of a polypeptide such as 95% "consistent" amino acid sequence The column is consistent with the query sequence, with the exception that for every 100 amino acids of the amino acid sequence, the target polypeptide sequence can include up to five amino acid changes. In other words, in order to obtain a polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence, up to 5% of the amino acid residues in the target sequence may be inserted, deleted, (inserted) or passed through another amino acid. Replace. Such alterations in the reference sequence may be present at the amino or carboxy terminal position of the reference amino acid sequence, or anywhere between their terminal positions, individually dispersed in a residue in the reference sequence, or in one or more The consecutive groups are dispersed within the reference sequence.

In fact, whether any particular polypeptide is associated with, for example, SEQ ID NO: an amino acid sequence of 2 (individual or a combination of a Factor IX moiety, an Fc moiety) or 4, or a known Factor IX or Fc polypeptide sequence of at least 85%, 90%, 95%, 96%, 97%, 98% Or 99% consistent can be determined by convention using known computer programs. A preferred method for determining the best overall match between a query sequence (reference sequence or original sequence) and the target sequence (also known as overall sequence alignment) can be used based on Brutlag et al., Comp. App. Biosci. The FASTDB computer program of the algorithm of 237-245 (1990) is used for the determination, which is incorporated herein by reference in its entirety. In sequence alignment, the query sequence and the target sequence are either nucleotide sequences or all amino acid sequences. The results of the overall sequence alignment are expressed as percent identity. The preferred parameters for use in the FASTDB amino acid alignment are: matrix = PAM 0, k-tuple = 2, mismatch penalty = 1, junction penalty = 20, randomization group length = 0, truncation score =1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the target amino acid sequence Degree, whichever is shorter.

If the target sequence is missing due to the N-terminus or C-terminus If it is missing internally and shorter than the query sequence, the results must be manually corrected. This is because the FASTDB program does not consider the N-terminal and C-terminal truncation of the target sequence when calculating the overall percent identity. For the sequence of the target sequence truncated at the N-terminus and the C-terminus, the N-terminus and the C-terminus of the target sequence are not associated with the corresponding target by calculating the percentage of the total base of the query sequence. The number of bases matched/aligned by the residues to correct the percent identity. Whether the residues match/align are determined by the FASTDB sequence alignment results. This percentage is then subtracted from the percentage of consistency calculated using the FASTDB program above the specified parameters to obtain a final consistency percentage score. This final consistency percentage is scored for the purpose of the present invention. For the purpose of manually adjusting the percent identity score, only the N-terminal and C-terminal residues of the target sequence that are not matched/aligned with the query sequence are considered. That is, only the position of the residue outside the farthest N-terminus and C-terminal residue of the target sequence is queried.

For example, the sequence of the 90 amino acid residues is A 100-residue query sequence is aligned to determine the percent identity. The deletion is present at the N-terminus of the target sequence, so the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent the 10% sequence (the number of unmatched residues at the N-terminus and the C-terminus / the total number of residues in the query sequence), so the score is subtracted from the percent identity calculated by the FASTDB program. Go to 10%. If the remaining 90 residues match exactly, the final percent identity will be 90%. In another example, 90 residues will be The target sequence is compared to a 100 residue query sequence. This time, the deletion is an internal deletion, so there are no residues at the N-terminus or C-terminus of the target sequence that are not matched/aligned with the query sequence. In this case, the percent consistency calculated by FASTDB is not manually corrected. Again, only the residue positions that are not matched/aligned with the query sequence outside the N-terminus and C-terminus of the target sequence are manually corrected, as shown in the FASTDB alignment. For the purposes of the present invention, no other manual corrections will be made.

Polynucleotide variants can be in the coding region, non-coding region or two There are changes in the person. Particularly preferred are polynucleotide variants which contain a silent substitution, addition or deletion, but which do not alter the property or activity of the encoded polypeptide. Nucleotide variants produced by silent substitution due to degeneracy of the genetic code are preferred. Further, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted or added in any combination are also preferred. Polynucleotide variants can be optimized for a variety of reasons, for example, to optimize codon expression in a particular host (to make codons in human mRNA a preferred codon for a bacterial host such as E. coli) And produced.

Naturally occurring variants are called "dual gene variants" And refers to one of several alternative forms of the gene occupying a given locus on the chromosome of an organism (Genes II, Lewin, B. ed., John Wiley & Sons, New York (1985)). Such dual gene variants can vary at the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants can be produced by mutation induction techniques or by direct synthesis.

Variants can be produced to modify or alter the characteristics of a polypeptide using known methods of protein engineering and recombinant DNA techniques. For example, One or more amino acids are deleted at the N-terminus or C-terminus of the autocrine protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), which is hereby incorporated by reference in its entirety, are hereby incorporated by reference to the disclosure of the third, the A variant KGF protein that still has heparin binding activity. Similarly, this protein exhibits up to a tenfold increase in activity after deletion of 8-10 amino acid residues from the carboxy terminus of interferon gamma. (Dobeli et al, J. Biotechnology 7: 199-216 (1988), which is incorporated herein by reference in its entirety.)

In addition, there is sufficient evidence that variants often retain biological activity similar to the biological activity of naturally occurring proteins. For example, Gayle and coworkers (J. Biol. Chem. 268: 22105-22111 (1993), which is incorporated herein by reference in its entirety), have been subjected to extensive mutational analysis of the human cytokine IL-1a. It uses random mutation induction to generate more than 3,500 individual IL-1a mutants, each with an average of 2.5 amino acid changes over the total length of the molecule. Multiple mutations at each possible amino acid position were examined. The researchers found that "[most] molecules can be altered with minimal impact on [binding or biological activity]." (See Abstract.) In fact, of the 3,500 or more nucleotide sequences examined, only 23 unique amino acid sequences produced significantly different activities than wild-type proteins.

As mentioned above, polypeptide variants include modified polypeptides. Modifications include acetylation, deuteration, ADP-ribosylation, guanidine, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of nucleotides or nucleotide derivatives, Valence linking lipids or lipid derivatives, covalently linking phospholipid inositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation Valence cross-linking, formation of cysteine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, PEGylation, proteolytic treatment, phosphorylation, prenylation, racemization, selenization, sulfation, transfer-RNA-mediated addition of amino acids (such as spermine deuteration) to proteins and ubiquitin Chemical.

IV. Pharmaceutical Composition

The long-acting FIX polypeptide can be formulated into a pharmaceutical composition. The pharmaceutical composition can be formulated for administration to humans. The pharmaceutical compositions for use in the methods of the invention comprise a pharmaceutically acceptable carrier, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Phosphate), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated plant fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, Zinc salt, colloidal cerium oxide, magnesium tricaprate, polyvinylpyrrolidone, cellulose-based material, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene Segment polymer, polyethylene glycol and lanolin. Various methods of formulation of the present invention are well known in the art.

Long-acting FIX polypeptides can be formulated into pharmaceutical compositions or formulations. In certain formulations provided herein, the long acting FIX polypeptide is formulated into a sterile, preservative, pyrogen free, lyophilized white to off-white powder for intravenous (IV) administration to the cake. Formulations are available in single use vials. Certain exemplary formulations of long acting FIX polypeptides are also known as eftrenonacog alfa.

In certain embodiments, a long acting FIX polypeptide (eg, The rFIXFc) formulation is provided in a single use vial which is manufactured to contain about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, about 400 IU/ml, or about 600 IU/ml after reconstitution with an appropriate amount of diluent. Long-acting FIX polypeptide. In certain embodiments in which the diluent is added until a final volume of about 5 ml, the single use vial can be rated to contain about 250, about 500, about 1000, about 2000, or about 3000 international units (IU) of long acting FIX polypeptide (eg, rFIXFc).

In certain embodiments, the rFIXFc polypeptide comprises and SEQ ID NO: Amino acid of 1 to 642 is at least 90%, 95% or 100% identical amino acid sequence.

In certain embodiments, in addition to an active long acting FIX polypeptide (eg, In addition to rFIXFc), the formulation also includes: sucrose (which can act as a stabilizer or accumulator), mannitol (which can act as a stabilizer or accumulator), L-histamine (which can act as a buffer) And polysorbate 20 or polysorbate 80 (which can act as a stabilizer). The formulation is provided with a diluent comprising a sterile sodium chloride solution. In certain embodiments, the diluent is provided in a pre-filled syringe.

Therefore, this article provides a long-acting FIX containing a specified amount. A pharmaceutical composition of a polypeptide (e.g., rFIXFc) (in IU) and an excipient of sucrose, mannitol, L-histamine, NaCl, and polysorbate 20 or polysorbate 80. The compositions provided herein contain various excipients at various concentrations, and the concentrations can be expressed in a variety of ways. For example, the concentration of a given excipient can be expressed as a molar concentration (eg, M or mM), weight/volume percent Ratio (for example, grams per 100 ml of diluent) or milligrams per milliliter (mg/ml). The formulations provided herein may contain a specified amount of each excipient with a certain degree of precision, which is within an approximate range, for example, the concentration is expressed only to a significant number (eg, about 0.1% (w/v)), or More precisely, for example up to 2, 3, 4, 5 or 6 significant figures (for example about 3.88 mg/ml with an accuracy of up to three significant figures). The degree of precision necessary may vary, for example, from the requirements of the established regulatory agency or the method of manufacture. In certain embodiments, the pharmaceutical compositions comprise a reconstituted formulation that can be provided in the form of a lyophilizate, optionally with a diluent.

In certain embodiments, the pharmaceutical composition comprises about 25 From IU/ml to about 1200 IU/ml rFIXFc, such as 50 IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml or 600 IU/ml long acting FIX polypeptide (e.g., rFIXFc). In certain embodiments, the pharmaceutical composition comprises about 3.88 mg/ml or about 25 mM L-histamine, about 23.8 mg/ml or about 2.4% (w/v) mannitol, about 11.9 mg/ml or A formulation of about 1.2% (w/v) sucrose, about 0.10 mg/ml or about 0.010% (w/v) polysorbate 20 or polysorbate 80 and about 3.25 mg/ml or about 55.6 mM NaCl. A 50 IU/ml, 100 IU/ml, 200 IU/ml or 400 IU/ml long acting FIX polypeptide (eg, rFIXFc) is included.

In certain embodiments, the pharmaceutical composition comprises about 5.43 Mg/ml or about 35 mM L-histamine, about 33.3 mg/ml or about 3.3% (w/v) mannitol, about 16.7 mg/ml or about 1.7% (w/v) sucrose, about 0.14 mg/ Mol or about 0.014% (w/v) polysorbate 20 or polysorbate 80 and a formulation of about 3.25 mg/ml or about 55.6 mM NaCl Contains 600 IU/ml long-acting FIX polypeptide (eg, rFIXFc).

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable An acceptable amount of sucrose. In certain embodiments, the pharmaceutical composition comprises from about 1% (w/v) to about 2% (w/v) sucrose, such as about 1.2% (w/v) sucrose or about 1.7% (w/v) sucrose. . In certain related embodiments, the pharmaceutical composition comprises from about 10 mg/ml to about 20 mg/ml sucrose, such as about 11.9 mg/ml sucrose or about 16.7 mg/ml sucrose.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable An acceptable amount of mannitol. In certain embodiments, the pharmaceutical compositions comprise from about 2% (w/v) to about 4% (w/v) mannitol, such as about 2.4% (w/v) mannitol or about 3.3% (w) /v) Mannitol. In certain related embodiments, the pharmaceutical compositions comprise from about 20 mg/ml to about 40 mg/ml mannitol, such as about 23.8 mg/ml mannitol or about 33.3 mg/ml mannitol.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable Acceptable amounts of both sucrose and mannitol. In certain embodiments, the pharmaceutical compositions comprise from about 1.0% to about 2.0% sucrose and from about 2.0% (w/v) to about 4.0% (w/v) mannitol, for example about 1.2% (w/v) Sucrose and about 2.4% (w/v) mannitol or about 1.7% (w/v) sucrose and about 3.3% (w/v) mannitol. In certain related embodiments, the pharmaceutical composition comprises from about 10 mg/ml to about 20 mg/ml sucrose and from about 20 mg/ml to about 40 mg/ml mannitol, such as about 11.9 mg/ml sucrose and about 23.8 mg/ml nectar. Sugar alcohol or about 16.7 mg/ml sucrose and about 33.3 mg/ml mannitol. In certain embodiments, sucrose and mannitol are provided as part of a lyophilizate which, after reconstitution with an appropriate amount of diluent, provides sucrose and sucrose at a desired concentration Sugar alcohol.

In certain embodiments, the pharmaceutical composition comprises about 50 mM With about 60 mM NaCl, for example about 55.6 mM NaCl. In certain related embodiments, the pharmaceutical composition comprises between about 3 mg/ml and about 4 mg/ml NaCl, such as about 3.25 mg/ml NaCl. In certain embodiments, NaCl is provided at a desired concentration in a diluent solution in which the lyophilizate comprising the long acting FIX polypeptide (eg, rFIXFc) is reconstituted.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable An acceptable amount of L-histamine. In certain embodiments, the pharmaceutical composition comprises between about 20 mM and about 40 mM L-histamine, such as about 25 mM L-histamine or about 35 mM L-histamine. In certain related embodiments, the pharmaceutical composition comprises between about 3 mg/ml and about 6 mg/ml of L-histamine, for example about 3.88 mg/ml L-histidine or about 5.43 mg/ml L-group. Amino acid. In certain embodiments, the L-histamine acid is provided as part of a lyophilizate which, upon reconstitution with an appropriate amount of diluent, provides L-histamine at a desired concentration.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable An acceptable amount of polysorbate 20 or polysorbate 80 is acceptable. In certain related embodiments, the pharmaceutical composition comprises between about 0.008% (w/v) and about 0.020% (w/v) of polysorbate 20 or polysorbate 80, for example about 0.010% (w) /v) Polysorbate 20 or polysorbate 80 or about 0.014% (w/v) polysorbate 20 or polysorbate 80. In certain related embodiments, the pharmaceutical composition comprises between about 0.08 mg/ml and about 0.2 mg/ml of polysorbate 20 or polysorbate 80, for example about 0.10% mg/ml. Sorbitol 20 or polysorbate 80 or about 0.14 mg/ml polysorbate 20 or polysorbate 80. In certain embodiments, polysorbate 20 or polysorbate 80 is provided as part of a lyophilizate which, upon reconstitution with an appropriate amount of diluent, provides polysorbate 20 or polysorbate at a desired concentration Ester 80.

In certain embodiments, the pharmaceutical composition comprises: about 25 Long acting FIX polypeptide (e.g., rFIXFc) between about IU/ml and about 700 IU/ml; sucrose between about 1% (w/v) and about 2% (w/v); about 2% (w/v) And about 4% (w/v) of mannitol; between about 50 mM and about 60 mM NaCl; between about 20 mM and about 40 mM of L-histamine; and about 0.008% (w/v) and About 0.015% of polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical compositions are provided in the form of a lyophilizate and a diluent. In certain embodiments, the amount of lyophilizate provides about 5 ml of a pharmaceutical composition having the desired concentration of the desired ingredient.

In certain embodiments, the pharmaceutical composition comprises: about 25 a long acting FIX polypeptide (e.g., rFIXFc) between about IU/ml and about 700 IU/ml; sucrose between about 10 mg/ml and about 20 mg/ml; mannitol between about 20 mg/ml and about 40 mg/ml; About 3 mg/ml and about 4 mg/ml of NaCl; about 3 mg/ml and about 6 mg/ml of L-histamine; and about 0.08 mg/ml and about 0.15 mg/ml of polysorbate Ester 20 or polysorbate 80. In certain embodiments, the pharmaceutical compositions are provided in the form of a lyophilizate and a diluent. In certain embodiments, the amount of lyophilizate provides about 5 ml of a pharmaceutical composition having the desired concentration of the desired ingredient.

Exemplary compositions are provided in Tables 1 and 2 of the Examples.

For example, the present disclosure provides a pharmaceutical composition, It comprises: about 50 IU/ml long-acting FIX polypeptide (eg rFIXFc); about 1.2% (w/v) sucrose; about 2.4% (w/v) mannitol; about 55.6 mM NaCl; about 25 mM L-histamine And about 0.010% (w/v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 100 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 1.2% (w/v) sucrose; about 2.4% (w/v) mannitol; about 55.6 mM NaCl; about 25 mM L-histamine; and about 0.010% (w/v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 200 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 1.2% (w/v) sucrose; about 2.4% (w/v) mannitol; about 55.6 mM NaCl; about 25 mM L-histamine; and about 0.010% (w/v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 400 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 1.2% (w/v) sucrose; about 2.4% (w/v) mannitol; about 55.6 mM NaCl; about 25 mM L-histamine; and about 0.010% (w/v) polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 600 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 1.7% (w/v) sucrose; about 3.3% (w/v) mannitol; about 55.6 mM NaCl; about 35 mM L-histamine; and about 0.014% (w/v) polysorbate 20 or polysorbate 80.

The present disclosure further provides a pharmaceutical composition, which comprises Containing: about 50 IU/ml long-acting FIX polypeptide (for example, rFIXFc); about 11.9 mg/ml sucrose; about 23.8 mg/ml mannitol; about 3.25 mg/ml NaCl; about 3.88 mg/ml L-histamine; and about 0.10 mg/ml polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 100 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 11.9 mg/ml sucrose; about 23.8 mg/ml mannitol; about 3.25 mg/ml NaCl; 3.88 mg/ml L-histamine; and about 0.10 mg/ml polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 200 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 11.9 mg/ml sucrose; about 23.8 mg/ml mannitol; about 3.25 mg/ml NaCl; 3.88 mg/ml L-histamine; and about 0.10 mg/ml polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 400 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 11.9 mg/ml sucrose; about 23.8 mg/ml mannitol; about 3.25 mg/ml NaCl; 3.88 mg/ml L-histamine; and about 0.10 mg/ml polysorbate 20 or polysorbate 80. The present disclosure further provides a pharmaceutical composition comprising: about 600 IU/ml long-acting FIX polypeptide (eg, rFIXFc); about 16.7 mg/ml sucrose; about 33.3 mg/ml mannitol; about 3.25 mg/ml NaCl; 5.43 mg/ml L-histamine; and about 0.14 mg/ml polysorbate 20 or polysorbate 80.

The present disclosure also provides components of a medical kit. The kit Includes one or more containers and accessories as appropriate. A kit as provided herein facilitates administration of an effective amount of a long acting FIX polypeptide (e.g., rFIXFc) to a subject in need thereof. In certain embodiments, the kit facilitates administration of a long acting FIX polypeptide (eg, rFIXFc) via intravenous infusion. In some embodiments, The kit facilitates self-administered long-acting FIX polypeptides (eg, rFIXFc) via intravenous infusion.

In certain embodiments, the present disclosure provides a medical kit a kit comprising: a first container comprising a lyophilized powder or cake, wherein the powder or cake comprises: (i) a long-acting FIX polypeptide (eg, rFIXFc), (ii) sucrose; (iii) mannitol; (iv) L-histidine; and (v) polysorbate 20 or polysorbate 80; and a 0.325% (w/v) NaCl diluent solution comprising a combination of the lyophilized powder of the first container Two containers. In certain embodiments, sufficient diluent is provided to produce about 5 ml of a formulation of a long acting FIX polypeptide (eg, rFIXFc) having the desired properties as disclosed herein. In certain embodiments, the second container is a pre-filled syringe with a plunger to allow for the addition of diluent to the first container, restoring the contents of the first container and transferring back to the syringe. In certain embodiments, the kit further provides a fitting for attaching the syringe to the first container. In certain embodiments, the kit further provides a needle and an infusion tube that will be coupled to a syringe containing a formulation that reconstitutes the long acting FIX polypeptide (eg, rFIXFc) to allow intravenous infusion of the formulation.

In certain embodiments, from about 200 IU to about 4000 Long acting FIX polypeptides (e.g., rFIXFc) are provided in a total amount of IU, e.g., about 250 IU, about 500 IU, about 1000 IU, about 2000 IU, or about 3000 IU.

In one embodiment, a medical kit is provided A first container comprising a lyophilized powder, wherein the powder comprises (i) about 250 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii) about 119 mg of mannitol; (iv) about 19.4 Mg L-histamine; and (v) about 0.50 mg polysorbate 20 or polysorbate 80; and included in the first The lyophilized powder of the container is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 50 IU/ml of a long acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; and (vi) about 0.01% (w/v) Polysorbate 20 or Polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 500 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol, (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 100 IU/ml long acting FIX polypeptide (eg, rFIXFc); (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% ( w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; and (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80 .

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 1000 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol, (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 200 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; About 25 mM L-histamine; and (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 2000 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 400 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% ( w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; and (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80 .

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 3000 IU of a long-acting FIX polypeptide (eg, rFIXFc), (ii) about 83.3 mg of sucrose; (iii) about 167 mg of mannitol; (iv) about 27.2 mg of L-histamine; and (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 600 IU/ml long acting FIX polypeptide (eg, rFIXFc); (ii) about 1.7% (w/v) sucrose; (iii) about 3.3% ( w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 35 mM L-histamine; and (vi) about 0.014% (w/v) polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 250 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol, (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 50 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannose Alcohol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mt. ml L-histamine and (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 500 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol, (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 100 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannose Alcohol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; and (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 1000 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol, (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 200 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannose Alcohol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; and (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 2000 IU of a long acting FIX polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose, (iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and when combined with the lyophilized powder of the first container, the volume is sufficient to produce a solution comprising a second container of 0.325% (w/v) NaCl: (i) about 400 IU/ml long-acting FIX polypeptide (eg, rFIXFc); (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannose Alcohol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; and (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

In another embodiment, a medical kit is provided, the kit A first container comprising a lyophilized powder, wherein the powder comprises: (i) about 3000 IU long acting FIX polypeptide (eg rFIXFc), (ii) about 83.3 mg sucrose; (iii) about 167 mg mannitol; (iv) about 27.2 mg L-histamine; and (v) about 0.7 mg polysorbate 20 or polysorbate 80; and a second container comprising, in combination with the lyophilized powder of the first container, in a volume sufficient to produce 0.325% (w/v) NaCl comprising: (i) about 600 IU /ml long-acting FIX polypeptide (eg rFIXFc); (ii) about 16.7 mg/ml sucrose; (iii) about 33.3 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 5.43 mg/ Ml L-histamine; and (vi) about 0.14 mg/ml polysorbate 20 or polysorbate 80.

In certain embodiments, the number of medical kits provided herein A container is a glass vial comprising a rubber stopper. In certain embodiments, the second container of the medical kit provided herein is a syringe body with a plunger. In certain embodiments, the syringe is a pre-filled syringe containing a diluent. In certain embodiments, the medical kit provided herein further includes a fitting that connects the glass vial to the syringe body. In certain embodiments, the medical kits provided herein further comprise an infusion tube with a needle adapted to be connected to the syringe for intravenous infusion.

In certain embodiments, a long acting FIX polypeptide (eg, The desired dose of rFIXFc) can be achieved via the use of a medical kit as provided herein. In certain embodiments, more than one medical kit can be used to achieve the desired dose. Provided herein is a method of combining or combining two or more formulations contained in a medical kit as provided herein to achieve a desired dosage.

In some embodiments, the pharmaceutical composition further comprises a short A FIX polypeptide. The short acting FIX polypeptide may comprise or consist of wild type FIX. Non-limiting examples of short acting FIX polypeptides include BENEFIX, MONONINE or ALPHANINE.

The pharmaceutical composition of the invention can be formulated into a liquid formulation, Freeze dry powder or suspension. The container containing the pharmaceutical composition can be a vial, cartridge or syringe. In a particular embodiment, the syringe comprising the pharmaceutical composition is a dual chamber syringe.

In certain embodiments, the pharmaceutical compositions of the invention are either frozen The reconstituted solution of the dry powder contains a preservative in an amount sufficient to provide antimicrobial activity. Pharmaceutically acceptable preservatives suitable for use in pharmaceutical compositions are well known in the art. For example, examples of pharmaceutically acceptable preservatives include, but are not limited to, phenol, m-cresol, benzyl alcohol, chlorobutanol, methyl paraben, propyl paraben, phenoxyethanol , any other pharmaceutically acceptable preservative, and any combination thereof. In a particular embodiment, the preservative is benzyl alcohol. In some embodiments, the pharmaceutical composition comprises benzyl alcohol at a concentration between 0.5% and 0.9%.

The invention has been described in detail, by reference to the following examples The invention is more clearly understood and is not intended to limit the invention. All patents and publications mentioned herein are expressly incorporated herein by reference.

V. Preparation method

Can be used in a host containing a vector encoding a long-acting FIX polypeptide Long acting FIX polypeptides are produced in cells. In one embodiment, the host cell comprises one or more first nucleotide sequences encoding a FIX polypeptide and a first FcRn polypeptide, a second nucleotide sequence encoding a second FcRn polypeptide, and optionally a protein invertase Vector transformation of the third nucleotide sequence (e.g., PC5). As used herein, a expression vector refers to any nucleic acid that, when introduced into a suitable host cell, is required for insertion into the transcription and translation of the coding sequence, or in the case of an RNA viral vector, is required for replication and translation. Construct body. Expression vectors can include plastids, phagemids, viruses, and derivatives thereof.

Gene expression control sequences as used herein to assist Any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, that is operably linked to efficient transcription and translation of the encoding nucleic acid. The gene expression control sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other components Sex promoter. Exemplary viral promoters that constitutively function in eukaryotic cells include, for example, from cell giant virus (CMV), simian virus (eg, SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), and lous Rous sarcoma virus, cell giant virus, long terminal repeat (LTR) of Moloney leukemia virus and other retroviral promoters, and thymidine kinase promoter of herpes simplex virus child. Other constitutive promoters are known to those of ordinary skill in the art. Promoters suitable for use as gene expression sequences of the present invention are also included Including an inducible promoter. Inducible promoters are expressed in the presence of an inducing agent. For example, a metallothionein promoter is induced to facilitate transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

Examples of vectors include, but are not limited to, viral vectors or plastids body. Platinum carriers are widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. In the past few years, plastid vectors have been found to be particularly advantageous for the delivery of genes to cells in vivo due to their inability to replicate within the host genome and integrate into the host genome. However, such plastids having a promoter compatible with the host cell can express the peptide from a gene operably encoded in the plastid. Some of the commonly used plastids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plastids, pRC/CMV, various pCMV plastids, pSV40 and pBlueScript. Other examples of specific plastids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plastids are well known to those of ordinary skill in the art. In addition, standard molecular biology techniques can be used to custom design plastids to remove and/or add specific DNA fragments.

One or more expression vectors are then transfected or co-transfected into The polypeptide is expressed in a suitable cell. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al., (1978) Cell 14: 725), electroporation (Neumann et al. (1982) EMBO J 1: 841) and liposome-based reagents. A variety of host expression vector systems are available for the expression of the proteins described herein, including both prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA or plastid DNA containing appropriate coding sequences (eg, E. coli); yeast transformed with recombinant yeast or fungi expressing the appropriate coding sequence or a filamentous fungus; an insect cell system infected with a recombinant viral expression vector (eg, a baculovirus) containing a suitable coding sequence; and a recombinant viral expression vector (eg, cauliflower mosaic virus or tobacco mosaic virus (tobacco mosaic) Virus)) a plant cell system that is transformed or transformed with a recombinant plastid expression vector (eg, Ti plastid) containing the appropriate coding sequence; or an animal cell system, including mammalian cells (eg, HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. Such as As used herein, a eukaryotic cell refers to any animal or plant cell having a pronucleus of the endosperm. Eukaryotic cells of animals include cells of vertebrate (e.g., mammals) and cells of invertebrates (e.g., insects). Plant eukaryotic cells may include, without limitation, yeast cells. Eukaryotic cells differ from prokaryotic cells such as bacteria.

In certain embodiments, the eukaryotic cell is a mammalian Cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell Strain. HEK 293 cells can be derived from CRL-1533 from the American Type Culture Collection of Manassas, VA and from 293-H cells (catalog number 11631-017) or 293-F cells (catalog number 11625-019). Invitrogen (Carlsbad, Calif.) obtained. In some embodiments, the mammalian cell is a PER.C6® cell, which is a human cell line derived from the retina. PER.C6® cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from the American Type Culture Collection (Manassas, VA.) (e.g., CHO-K1; CCL-61). In other embodiments, the mammalian cell is a young hamster kidney (BHK) cell. BHK cells are available from the American Type Culture Collection (Manassas, Va.) (eg, CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell that is a hybrid cell line of HEK293 cells and a human B cell line. Mei et al, Mol. Biotechnol. 34(2): 165-78 (2006).

The method can further comprise a purification step. In this technology Various known purification steps are well known.

VI. Methods, systems, and storage media for estimating individualized drug delivery information, patient personalized PK information, and patient median PK information

The invention also includes a method of estimating rFIXFc administration information for individualization of a patient, the method comprising: (a) a pharmacokinetic (popPK) model comprising the rFIXFc population of Example 6, such as Table 21, and an optionally Bayesian estimation program Computer-based system receiving patient funds (b) using the popPK model, the Bayesian estimation program selected as appropriate, and the information received, using the computer-based system to calculate individualized rFIXFc dosing information, and (c) The personalized medication information is output by the computer based system.

In some embodiments, the method also includes selecting a dosing regimen based on the individualized dosing information of the output of (c) and administering rFIXFc to the patient according to the selected dosing regimen.

In some embodiments, the information to be treated is that the level of plasma FIX activity is increased after administration and the output information is the dose for acute treatment.

In some embodiments, the desired treatment outcome information is the desired dosing interval and the output information is the dose for control.

In some embodiments, the information to be treated is the desired dose and the output information is the interval for control.

The invention also includes a method for estimating a rFIXFc dosing regimen based on a median popPK, the method comprising: (a) receiving patient information by a computer-based system comprising the rFIXFc popPK model of Example 6, such as Table 21, and the Bayesian estimation program as appropriate. And at least one of the information to be treated, (b) using the popPK model, the Bayesian estimation program selected as appropriate, and the information received, calculating the median rFIXFc PK information by the computer-based system, and (c) The computer based system outputs the median PK information.

In some embodiments, the method also includes selecting a dosing regimen based on the median PK information of the output of (c), and administering to the patient according to the selected dosing regimen The person is administered rFIXFc.

The invention also includes an estimate of individual patient rFIXFc PK The method comprises: (a) receiving individual rFIXFc PK information by a computer-based system comprising the rFIXFc population pharmacokinetic (popPK) model of Example 6 and the Bayesian estimation program, and (b) using popPK The model, the Bayesian estimation program selected as appropriate, and the received information, the computerized system estimates the individualized patient rFIXFc PK information, and (c) the personalized patient PK information is output by the computer based system.

In some embodiments, the method also includes output based on (c) The individualized patient PK information selects the dosing regimen and the patient is administered rFIXFc according to the selected regimen.

In some embodiments, (a) further includes The brain system receives patient information.

In some embodiments, patient information is age (eg, 12 Years old and older) or weight. Other patient information included diagnostic (baseline) FIX content, PK measurements, PK sampling time, dosing history (if PK samples were taken from multiple doses), actual dose, FIX activity level, and the like.

In some embodiments, the information to be treated is, for example, The desired PK or desired result, for example, the degree of plasma FIX activity after the dose is increased, the desired dosing interval, and the desired dose.

In some embodiments, the output information is, for example, a PK curve, PK parameters such as incremental recovery (Cmax/dose), mean residence time, terminal t1/2, clearance, Vss, AUC/dose, dose and associated trough, and Interval and related valley bottom.

For example, to evaluate individualized patient PK, the system can It is recommended that the user enter 2-3 optimized PK sampling time points. In this case, the system output may include a PK curve and one or more selected PK parameters, such as incremental recovery (Cmax/dose), average residence time, terminal t1/2, clearance, Vss, AUC, and up to 1%. Or X% of the time, etc. For example, Figure 15.

As another example, to use the loss discussed in the previous paragraph Individual PK parameters are selected for individualized dosing regimens, (i) the dose selected for acute therapy can be increased based on the degree of plasma FIX activity entered by the user after the dose, and (ii) the dose selected for control can be selected. The selected interval based on the user's input, or (iii) the selected interval for control, may be based on the desired dose entered by the user. In the first case, the system can output a dose (IU) based on the patient's incremental recovery. For example, Figure 16. In the second case, the system output can be a table of doses and associated troughs (eg, x IU/kg, 1% trough, y IU/kg, 2% trough, etc.). For example, the upper part of Figure 17. In the third case, the system output can be a table of intervals and associated valleys (e.g., x day, 1% valley, y IU/kg, 2% valley, etc.), such as the lower portion of Figure 17.

Users may wish to enter no individualized PK data Use the system below. In this case, the drug delivery output will be individualized based on the median population rather than on a particular patient. For example, Figure 18. In this manner, the user enters, for example, body weight and age, and (i) the degree of plasma FIX activity is increased after the dose, (ii) the desired dosage interval for control, or (iii) the desired dose for control. In the first case, the system can output a dose. in In the second case, the system can output dose and associated troughs. See Table 16. In the third case, the system can output the interval and the associated valley. See Table 16.

In some embodiments, the system complies with patient privacy laws. In some embodiments, the system is encrypted, for example, with SSL. In some embodiments, inputting patient information is generated anonymously.

In some embodiments, the system includes a user assistance function.

The method can be performed by, for example, a physician, a nurse, or another health care practitioner.

Other embodiments include a computer readable storage medium storing instructions that when executed by a processor cause the processor to perform any of the above methods.

Other embodiments include a system including a processor and a memory having stored thereon instructions that, when executed by the processor, cause the processor to perform any of the above methods.

The user of the system or computer readable storage medium can be, for example, a physician, a nurse or another health care practitioner.

For other embodiments of these aspects of the invention, see Examples 5, 6 and 8 and the figures discussed therein.

VII. Example Computing Environment

The various modeling techniques, dose calculations, and estimates described herein can be implemented by software, firmware, hardware, or a combination thereof. Figure 19 illustrates An example computer system 1900 in which embodiments or portions thereof can be implemented in computer readable code form. For example, the modeling of Examples 5 and 6 and/or the patient treatment simulation of Example 8 can be implemented in system 1900.

Computer system 1900 includes one or more processors, such as Processor 1904. The processor 1904 is coupled to a communication infrastructure 1906 (e.g., a bus or network).

The computer system 1900 also includes a main memory 1908, preferably The machine accesses memory (RAM) and may also include auxiliary memory 1910. According to an implementation, the user interface material can be stored, for example, and not limited to, in the main memory 1908. Main memory 1908 can include, for example, cache memory and/or static and/or dynamic RAM. The auxiliary memory 1910 may include, for example, a hard disk drive and/or a portable storage drive machine. The portable storage drive unit 1914 may include a floppy disk drive, a tape drive, a disc drive, a flash memory, or the like. The removable storage drive 1914 reads and/or writes the removable storage unit 1916 in a well known manner. The removable storage unit 1916 can include floppy disks, magnetic tapes, optical disks, and the like that are read and written by the removable storage drive machine 1914. As will be appreciated by those skilled in the art, the removable storage unit 1916 includes a computer readable storage medium having stored computer software and/or data.

Computer system 1900 can also include a display interface 1902. display Interface 1902 can be adapted to communicate with display unit 1930. Display unit 1930 can include a computer monitor or similar device for displaying graphics, text, and other materials received via communication infrastructure 1906 autonomous memory 1908. In an alternative implementation, the auxiliary memory 1910 can include Computer programs or other instructions are loaded into other similar facilities in computer system 1900. Such facilities may include, for example, a removable storage unit 1922 and an interface 1920. Examples of such facilities may include a box and box interface (such as found in video game devices), removable memory chips (such as EPROM or PROM) and associated sockets, and software and data self-removable storage units. 1922 is transferred to other removable storage unit 1922 and interface 1920 of computer system 1900.

Computer system 1900 can also include a communication interface 1924. communication Interface 1924 allows software and data to be transferred between computer system 1900 and external devices. The communication interface 1924 can include a data machine, a network interface (such as an Ethernet card), a communication port, a PCMCIA slot and card, or the like. The software and data transferred via the communication interface 1924 are in the form of signals, which may be electronic signals, electromagnetic signals, optical signals, or other signals that can be received by the communication interface 1924. These signals are provided to communication interface 1924 via communication path 1926. Communication path 1926 carries signals and can be implemented using wires or cables, fiber optics, telephone lines, mobile phone links, RF links, or other communication channels.

In this document, the term "computer readable storage medium" is used. Commonly used to refer to non-transitory storage media, such as removable storage unit 1916, removable storage unit 1922, and hard disk mounted in hard disk drive 1912. A computer readable storage medium may also refer to one or more types of memory, such as main memory 1908 and auxiliary memory 1910, which may be a memory semiconductor (eg, DRAM, etc.). These computer program products are facilities for providing software to the computer system 1900.

Computer programs (also known as computer control logic) are stored in the main memory Body 1908 and/or auxiliary memory 1910. The computer program can also be received via communication interface 1924 and stored on main memory 1908 and/or auxiliary memory 1910. These computer programs, when executed, enable computer system 1900 to implement embodiments as discussed herein. In particular, the computer program, when executed, enables the processor 1904 to implement the methods of the present disclosure, such as some of the methods discussed above. Therefore, such computer programs represent controllers of computer system 1900. When the embodiment uses software, the software can be stored in a computer program product and loaded into computer system 1900 using removable storage drive 1914, interface 1920, or hard drive 1912.

Embodiments may be related to storage in any computer readable medium A computer program product of software on the body. The software, when executed in one or more data processing devices, causes the data processing device to operate as described herein. Embodiments may employ any computer usable or readable medium. Examples of computer readable storage media include, but are not limited to, non-transitory primary storage devices (eg, any type of random access memory) and non-transitory secondary storage devices (eg, hard drive, floppy disk, CD ROMS, ZIP) Disks, magnetic tapes, magnetic storage devices and optical storage devices, MEMS, nanotechnology storage devices, etc.). Other computer readable media include communication media (eg, wired and wireless communication networks, regional networks, wide area networks, internal networks, etc.).

Instance Example 1. Product Description

rFIXFc is a long-acting fully recombinant fusion protein consisting of human coagulation factor IX (FIX) covalently linked to the Fc domain of human immunoglobulin G1 (IgG1). The one-stage amino acid sequence of the Factor IX portion of rFIXFc is identical to the Thr ¹⁴⁸ dual gene form of plasma-derived Factor IX and has similar structural and functional characteristics to endogenous Factor IX. The Fc domain of rFIXFc contains the hinge region, CH2 region and CH3 region of IgG1. rFIXFc contains 869 amino acids with a molecular weight of approximately 98 kilodaltons.

rFIXFc has been widely used by recombinant DNA technology Produced in the human embryonic kidney (HEK) cell line. This cell line expresses rFIXFc to a defined cell culture medium free of any protein native to animal or human origin. rFIXFc was purified by a series of chromatography procedures that did not require the use of monoclonal antibodies. The method includes multiple viral clearance steps, including 15 nm virus cut-off filtration. No human or animal additives are used during cell culture, purification, and formulation.

rFIXFc belongs to the medical treatment group: anti-hemorrhagic agent, B02BD04. It is provided in a single-use vial with a sterile, preservative-free, pyrogen-free, lyophilized white to off-white powder to a cake in an intravenous (IV) formulation, accompanied by a liquid diluent in a pre-filled syringe. In addition to rFIXFc, the pharmaceutical composition also contains sucrose, L-histamine, mannitol, and polysorbate 20 in a lyophilizate, and a sodium chloride solution (0.325%) contained in a sterile solvent. Each single vial is rated to contain 250, 500, 1000, 2000 or 3000 International Units (IU) of rFIXFc. When reconstituted with the diluent provided, the product contained the following excipients at the concentrations shown in Table 1 or Table 2 below: sucrose, sodium chloride, L-histamine, mannitol, and polysorbate 20. The pharmaceutical composition is formulated for intravenous administration only after reconstitution.

Each package contains a plug (butyl) and a flip-type cover (aluminum) A powder vial (type 1 glass), 5 ml of solvent in a prefilled syringe (type 1 glass) with a plunger (butyl), tip (butyl) and sterile vial adapter recovery device.

Example 2: Provisioning method

rFIXFc drug is an injection intended for intravenous administration Sterile lyophilized powder. It is supplied in aseptically filled single use vials, each containing 250, 500, 1000, 2000 and 3000 IU. The vial was a 10 mL USP/Ph. Eur.1 glass vial sealed with a 20 mm Teflon coated butyl rubber lyophil plug and an aluminum flip type crimp cap. Prior to lyophilization, the 250 IU to 2000 IU vial has a nominal fill volume target of 5 mL and a 3000 IU vial has a nominal fill volume target of 7 mL. The composition of the formulated excipients prior to lyophilization was the same for all dose strengths. The powder for injection is reconstituted with 5 mL of diluent containing 0.325% (w/v) sodium chloride supplied in a sterile pre-filled syringe.

The composition of the drug solution before lyophilization is presented in Table 3. The composition of the lyophilized powder is presented in Table 4. The composition of the drug after recovery is presented in Table 1 or Table 2. (Example 1).

By connecting the syringe to a standard intravenous infusion tube/needle The group is administered intravenously by intravenous delivery of rFIXFc by standard methods known to those of ordinary skill in the art.

Example 3: Dosage and Method / Method of Calculating Initial Estimated Dose

rFIXFc is indicated for adults and children with hemophilia B (congenital factor IX deficiency) 12 years old) For long-acting anti-hemophilic factors (recombination) such as controlling and preventing bleeding events, routine prevention and treatment to prevent bleeding events or reducing the frequency of bleeding events, and perioperative management (surgical prevention).

Administration of the formulated rFIXFc as described in Example 1 can be estimated as described in the Examples, but can also be determined by standard tests, such as FIX activity assays described elsewhere herein.

It is expected that 1 IU of rFIXFc per kilogram of body weight will increase the circulating content of Factor IX by 1% [IU/dL]. rFIXFc has been shown to have a long circulating half-life.

It is usually not necessary to adjust the recovered dose. Because of the subject's medicine The kinetics (eg, half-life, in vivo recovery) and the clinical response to rFIXFc may vary, so the expected increase in factor IX content in vivo may be estimated as IU/dL (or % of normal value) using the following formula or Required dose: IU / dL (or % of normal value) = [total dose (IU) / body weight (kg)] × recovery (IU / dL per IU / kg) or dose (IU) = weight (kg) ×The desired factor IX is increased (% of IU/dL or normal value) × the reciprocal of the recovery rate (IU/kg per IU/dL)

The following table (Table 5) can be used to guide the administration of bleeding events:

The subsequent therapeutic dose and duration will depend on the individual clinical response, the severity of Factor IX deficiency, and the location and extent of bleeding (see pharmacokinetics in Example 5 below).

The following table (Table 6) can be used to guide the administration of perioperative management (surgical control):

For routine control, the recommended starting plan is: once a week 50 IU/kg or 100 IU/kg once every 10-14 days. Either regimen can be adjusted based on the subject response (see pharmacokinetics of Example 5 below).

rFIXFc is contraindicated for the appearance of products or their components Among subjects with hypersensitivity reactions (including allergies).

The clinical response to rFIXFc can vary. If the bleeding is not pushed With dose control, the plasma level of Factor IX can be determined and a sufficient dose of rFIXFc can be administered to achieve a satisfactory clinical response. If the subject's plasma factor IX content does not increase as expected or if bleeding is not controlled after administration of rFIXFc, the subject's plasma inhibitor can be tested The presence of (eg neutralizing antibodies). The production of Factor IX inhibitors in subjects using rFIXFc can be monitored by appropriate clinical observations and laboratory tests known to those of ordinary skill in the art.

When clinically indicated, for example, a single-stage coagulation The extent of Factor IX activity in the plasma of the subject was monitored to confirm that sufficient Factor IX content had been achieved and maintained. The production of Factor IX inhibitors in the plasma of the subject can be further monitored.

Example 4. Safety, efficacy, and improved pharmacokinetics shown in phase 3 clinical trials ("B-LONG" study) of recombinant Fc fusion factor IX with extended half-life B-LONG research design

The study was designed as a global open-label, non-randomized multicenter (50 research sites in 17 countries) Phase 3 study.

The aim was to evaluate the intravenous injection of recombinant Factor IX Fc fusion protein (rFIXFc) in the control and prevention of bleeding events, routine control and prevention in individuals with severe hemophilia B (pre-treated subjects (PTP)) / or efficacy, tolerance, pharmacokinetics (PK) and safety in perioperative management. Figure 1 shows a diagram of the study design. The study disclosed in this example is the largest registration test for any therapeutic agent for hemophilia B, with the most extensive PK analysis performed to date. The duration of the study was approximately 72 weeks.

Key inclusion criteria include (1) male, (2) greater than 12 years old (weight at least 40 kg), and (3) diagnosed as endogenous factor IX activity 2%( 2 IU/dL FIX: C) severe hemophilia B, (5) has the following history: 100 previous recorded exposure days (ED) of any currently marketed FIX product, bleeding events 12 weeks prior to enrollment and/or treatment with FIX, or 52 weeks prior to enrollment if they have received paroxysmal treatment At least 8 bleedings occurred, (6) will have an association with FIX or intravenous immunoglobulin-related inhibitors or allergies, other coagulopathy, uncontrolled HIV infection, renal dysfunction, or active severe liver disease Subjects were excluded from the study, and (7) excluded subjects who were unable or unwilling to comply with the control protocol or diary requirements or who had received immunosuppressive drugs prior to the study. 12 months of subjects.

The three treatment teams are team 1 (fixed weekly interval control), Team 2 (individualized interval prevention), team 3 (paroxysmal [on-demand] treatment), team 4 (perioperative management).

In team 1, the subject is treated weekly with an initial dose of 50 IU/kg, and then the dose is adjusted based on the PK profile to maintain a trough factor content sufficient to prevent bleeding to maintain 1-3 IU/dL above baseline at the bottom and/or If the subject experiences for 3 consecutive months Two doses of spontaneous bleeding were adjusted. A graph showing continuous PK subgroup administration and PK sampling of team 1 is shown in FIG. In particular, the rFIXFc dose was adjusted based on monitoring of the trough at weeks 4, 16, 26 and 39. If the pharmacokinetic value indicates that the patient's bottom content is above 3 IU/dl above baseline or higher at the end of the week, the dose is lowered to achieve a target of 1 IU/dl to 3 IU/dl above the baseline. If the pharmacokinetic value indicates that the patient's trough content will be less than 1 IU/dl above baseline at the end of the week, the dose is increased to achieve a target of 1 IU/dl to 3 IU/dl above the baseline. If the target range is not achieved, the plasma sample is manipulated to rule out the presence of the inhibitor. Duplicate trough content was achieved approximately one month after any dose adjustment and repeated monthly until no other adjustments were necessary. Only the dose of rFIXFc was adjusted in this treatment team.

In team 2, subjects were treated with 100 IU/kg at an initial interval of 10 days, and then the interval was adjusted to maintain a trough factor content sufficient to prevent bleeding (adjusted intervals according to the subject's PK profile to maintain high activity at the bottom of the valley) 1-3 IU/dL at baseline and/or if the subject experiences for 3 consecutive months 2 spontaneous bleeding is adjusted interval). Monitoring was conducted at weeks 4, 16, 26 and 39 to ensure that the FIX bottom content was maintained. Only the interval of rFIXFc was adjusted in this treatment team.

Unacceptable experience in Team 1 or Team 2 Any patient with blood, two or more spontaneous bleeding for 3 consecutive months, adjusted dose (team 1) or interval (team 2) to achieve FIX trough content above baseline 3IU/dl to 5IU/dl aims. If unacceptable bleeding does not occur at the target trough of rFIXFc above baseline 3 IU/dl to 5 IU/dl, then at the discretion of the investigator and after agreement with the promoter, rFIXFc can be adjusted to achieve 1 IU/dl to The target of 3IU/dl is the bottom.

In team 3, the subject received the need for bleeding rFIXFc paroxysmal treatment (eg 20 IU/kg to 100 IU/kg, depending on the severity of bleeding). Mild bleeding events were treated with 20-30 IU/kg, moderate to severe bleeding events were treated with 25-50 IU/kg, and severe to life-threatening bleeding events were treated with 50-100 IU/kg for patient-based pharmacokinetic profiles. Achieve 20-30%, 25-50%, and 50-100% of the bottom FIX content.

In team 4, 40-100 IU/kg rFIXFc was administered before and after major surgery, in which the dose was adjusted according to the type of surgery; Direct recruitment to the surgical team, followed by surgery to a treatment team (team 1, team 2 or team 3); or if surgery is required during the study, during the perioperative period Moved from another team to the surgery team.

Receiving rFIXFc in team 1 and team 3 as much as treatment 52 ± 1 week and team group 2 up to 50 exposure days (ED). Subjects in the previous prevention regimen only entered team 1 or team 2, while previous use of paroxysmal therapy could be recruited to any treatment team.

The continuous PK subgroup in team 1 received 50 IU/kg rFIX at baseline and subsequently eluted before the first dose of rFIXFc 5th. 120 hours after the PK evaluation of rFIX injection, subjects received 50 IU/kg rFIXFc and blood samples were collected over 240 hours for PK profiling. The rFIXFc PK profiling was repeated at week 26 for consecutive PK subgroups. All remaining subjects in the study also experienced discontinuous rFIXFc PK evaluation for 168-336 hours (7-14 days: team 1 = 10; team 2 = 14; team 3 = 7).

For the PK assessment, all subjects in all teams performed an initial PK assessment after their first dose of rFIXFc. A subset of subjects from team 1 was assigned to the consecutive PK subgroups assigned to the protocol to compare the PK of rFIXFc with recombinant Factor IX (rFIX, BeneFIX ^® ) as follows: after a 120 hour washout period, in team 1 the pharmacokinetic consecutive patients in the subgroup receiving 50IU / kg of recombinant FIX (rFIX, BeneFIX ^®) following the injection and subjected to 96 hours until the pharmacokinetics sampling: before the injection, since the injection start 10 (± 2) minutes, 1 hour ( ±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 (±2) hours, 72 (±3) hours, and 96 (±3) hours. Each patient must complete a minimum evaluable pharmacokinetic sampling up to a 72 hour time point for inclusion in the pharmacokinetic analysis group.

Complete rFIX pharmacokinetic sampling and start with the last rFIX dose After 5-day, the patient received a 50 IU/kg dose of rFIX Fc fusion protein (rFIXFc) while pharmacokinetic evaluation was performed over 240 hours. rFIXFc sampling was performed as follows: 10 (±2) minutes, 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 before injection. (±2) hours, 96 (±3) hours, 144 (±3) hours, 168 (±3) hours, 192 (±3) hours, and 240 (±3) hours. Each patient must complete a minimum evaluable pharmacokinetic sampling up to a 168 hour time point for inclusion in the pharmacokinetic analysis group. Repetitive pharmacokinetic evaluation of rFIXFc was also performed at week 26 using the same sampling time course.

In team 1 and 2, the efficacy period is in pharmacokinetics The date and time of the first dose control after the completion of the sampling period begins and ends with the last dose (used to prevent bleeding events) as recorded in the electronic case report form (eCRF) or electronic diary (eDiary). In team 1 (continuous pharmacokinetics subgroup), the efficacy period was interrupted for a period of repeated repetitive pharmacokinetics, and in teams 1 and 2, the efficacy period was interrupted for all surgical/rehabilitation periods (for major surgery and Both minor surgery). The period of efficacy lasts until the last dose before the repeated pharmacokinetic dose (for the prevention or treatment of bleeding events) or until the last dose before the start of the surgery/rehabilitation period (for the prevention or treatment of bleeding events), followed by pharmacokinetics or surgery / The next dose after the end of the rehabilitation period is restored.

In team 3, the efficacy period is taken at the last pharmacokinetics Start at 1 minute after the sample time and end on the date of the last contact or the date of the last electronic journal entry, whichever is later. The period of efficacy was interrupted 1 minute before the start of the surgery/rehabilitation period and restarted at 00:01 on the day after the end of the surgery/rehabilitation period.

FIX activity by a single-stage plasma (activated partial thromboplastin time [the aPTT]) measured coagulation analysis, the analysis of the human plasma samples rFIXFc, BeneFIX ^® system and human FIX verified. Using commercially available aPTT reagents (e.g. Trinity Biotech (Automatic the APTT [based activator and phospholipid mixture of silicon dioxide])), Precision BioLogic (CRYOCHECK TM factor IX- removal of plasma) and normal reference plasma as calibrator of (Precision BioLogic (LLOQ 1 IU/dl) FIX activity in a citrate-containing plasma sample having a potency relative to the World Health Organization's 3rd or 4th International Standard for Plasma, measured on a MDA 180 coagulation instrument. Accuracy is within 95% to 104% for all three FIX proteins, and intra-assay and inter-assay accuracy is typically within 10%.

Neutralizing antibodies were detected as follows: Nijmegen modified Bethesda analysis to detect neutralizing antibodies was performed at each visit during screening, baseline, and study treatment to monitor the production of inhibitors. After the first dose of rFIXFc, an inhibitor test was performed at the bottom of each of the scheduled clinical visits in teams 1 and 2, where the bottom was defined as the point after the longest interval between predetermined doses. Inhibitor testing was performed in team 3 after at least 72 hours (3 days) of elution. For new patients entering team 4, an inhibitor test was performed at screening and then a total of at least 4 EDs were achieved with rFIXFc within 4 weeks prior to the scheduled surgery. The formation inhibitor is defined as confirming the neutralizing antibody value in the second separate sample within 2-4 weeks. 0.6 Bethesda units (BU) / ml. Contrast intervals for inhibitor incidence were evaluated using the Clopper-Pearson binomial proportional-precision method.

Key efficacy outcome measures include (1) Teams 1, 2, and 3 Annual bleeding rate (ABR) (compared to each of the 2 control groups and the paroxysmal (on-demand) treatment group) (ie, (a) weekly control team (team 1) and paroxysmal treatment team ( Team 3) compare and (b) individualized interval control team (team 2) compared with paroxysmal team (team 3); (2) response to bleeding event treatment, use four points Hemorrhagic response scale (ie (a) consumption per subject; (b) weekly dose of team 1; (c) dosing interval for team 2; and (d) to prevent bleeding events The number of injections required and the dose per injection); and (3) the evaluation of the response of the subject to surgery with rFIXFc using a 4-point scale; the number of injections required to maintain hemostasis during the surgical period and Dose; estimated blood loss during surgery; and the number of infusions required for surgery. For annual bleeding rate measurements.

Pharmacokinetic (PK) results measures include the following: (1) PK profiles are assessed by user-defined 2-case model based on plasma FIX activity decay over time and validated for use in PK analysis, as evidenced by Single-stage (activated partial clotting hormone time) coagulation analysis in the central laboratory, and (2) PK of rFIXFc and recombinant factor IX (rFIX, BENEFIX ^® ) and a time of 1% above baseline.

Baseline (endogenous) for FIX active pharmacokinetic analysis FIX activity) is defined as the lowest observed FIX activity at the time of screening, before administration, after administration, or according to the patient's historical clinical record. For the lowest observation For patients with FIX activity below 1%, baseline FIX activity was set to zero; for patients with the lowest observed FIX activity between 1 IU/dl and 2 IU/dl, baseline FIX activity was set to the actual observed FIX value. The residual drug decays following a first order decay, where the rate of decay is determined individually. The user-specified and validated dual compartment model was used to analyze the profile of FIX activity over time corrected by baseline and residual drug. The user specification code automates the calculation of other secondary pharmacokinetic parameters (eg, 1% and 3% time), which are not included in the secondary parameter list of the WinNonlin library model (PHOENIX® WinNonlin 6.2.1.51; Pharsight). Therefore, the need to manually process data beyond the primary analysis is eliminated and the risk of introducing human error is minimized.

Key safety outcome measures include the following: (a) Laboratory values Clinically significant changes from baseline; (b) incidence of inhibitor production; and (c) incidence of adverse events (AE) that occurred beyond the perioperative management team (teams 1, 2, and 3 instead of 4) .

Statistical Analysis - Reporting the median annual bleeding rate (ABR) and making The estimated ABR was calculated using a negative binomial model that considered excessive dispersion to compare the ABR between team 1 and 2 (control regimen) and team 3 (paroxysmal therapy). Descriptive statistics were used to provide median and quartile range (IQR) values for each of team groups 1-3. The continuous PK assay population included team 1 subjects who received blood samples from the beginning of the injection to a minimum of 72 hours (for 50 IU/kg rFIX) and a minimum of 168 hours (for 50 IU/kg rFIXFc). An analysis of variance (ANOVA) using the study treatment (rFIX or rFIXFc) and subject variables was used and a 95% confidence interval for the geometric mean of each treatment was provided. Based on FDA guidance, the study has sufficient ability to detect the presence of inhibitors. Significance, the FDA guidelines for the emergence of inhibitors in clinical studies can use a binomial distribution of 2% 95% CI that produces a true inhibitor incidence (0.05% to 10.65%) using the precise Klopp-Pearson method Fully modeled. All statistical tests were performed on both sides at a significant 5% level. When a predetermined number of primary surgical procedures have been completed, the study is terminated after the interim analysis.

B-LONG results Subject

A total of 123 subjects were recruited in the study. 115 (93.5%) subjects completed the study (3 agreed to withdraw; 1 was lost to follow; 2 were withdrawn due to a violation of the program; and 2 were suspended due to an unacceptable event). Subjects were subjected to a 96-hour washout period prior to initial study dosing, at which time a PK assessment was performed. Subjects recruited were enrolled in one of the four treatment teams detailed above (see also Figure 1).

- Team 1 (weekly prevention), n=63 (59 completed)

- Team 2 (individualized interval control), n=29 (27 completed)

- Team 3 (paroxysmal treatment), n=27 (26 completed)

- Team 4 (perioperative management), n=12 (3 completed), 14 operations (8 of 12 subjects also participated in other treatment teams (teams 1, 2 and 3))

The baseline characteristics of the study subjects (see Table 7) showed a substantial difference in the population of human immunodeficiency virus and hepatitis C virus infection, reflecting the general hemophilia B population. The genotype profile is consistent with the expected genotype profile in the study population (55% with missense mutations).

>80% of weekly infusions in 48 subjects in previous control regimens 2 times. Subjects in the control group (team groups 1 and 2) were well balanced in terms of previous protocols and bleeding history. There were fewer subjects with a target joint in team 2 and more subjects with a baseline FIX activity < 1 IU/dL in team 3. The median (minimum, maximum) treatment duration for team groups 1, 2, and 3 was 51.6 (<1,97), 58.3 (<1,126), and 40.9 (28,54) weeks, respectively, and the median (minimum, maximum) The exposure days were 55.0 (1,105), 38.0 (1,71), and 16.0 (4,35), respectively. A total of 5243 rFIXFc injections were administered during this study, corresponding to 5144 EDs (117.1 patient exposure years). The treatment adherence of 80% of subjects who were compliant with their assigned dose (team 1) or dosing interval (team 2) was 96.6% overall in the control team (team 1 = 95.1%, team 2 = 100%).

For patients in team 4, major surgery is defined as usual but Any surgical procedure (optional or urgent) that does not necessarily involve general anesthesia and/or respiratory assistance, which penetrates and exposes the main body cavity, or causes substantial damage to the body or physiological function (eg laparotomy, thoracotomy, opening) Craniotomy, joint replacement or amputation).

Study visit time schedule:

For team groups 1 to 3, study visits occurred at screening (8 weeks), baseline, week 4, week 16, week 26, week 39, and week 52. In addition, patients in teams 1 through 3 received a 30-day follow-up call unless they were enrolled in an ongoing extension study (NCT01425723). Patients in team 4 underwent a study visit at screening, baseline, day of surgery, 1 week after surgery, and 1 week after rehabilitation.

Continuous pharmacokinetic subgroup

The sampling duration of rFIX and rFIXFc is based on the previously reported half-life, allowing the decay time (usually three to five times the previously observed half-life) to be sufficient to accurately describe the pharmacokinetics. After a 120 hour washout period, patients in the continuous pharmacokinetic subgroup of team 1 received an injection of 50 IU/kg recombinant FIX (rFIX) and were subjected to pharmacokinetic sampling as follows until 96 hours: before injection, from the start of injection 10 (±2) minutes, 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 (±2) hours, 72 (±3) hours And 96 (±3) hours. Each patient must complete a minimum evaluable pharmacokinetic sampling up to a 72 hour time point for inclusion in the pharmacokinetic analysis group.

Complete rFIX pharmacokinetic sampling and start with the last rFIX dose After 5 days, the patient received a dose of 50 IU/kg of rFIX Fc fusion protein (rFIXFc) while pharmacokinetic evaluation was performed over 240 hours. rFIXFc sampling was performed as follows: 10 (±2) minutes, 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 24 (±2) hours, 48 before injection. (±2) hours, 96 (±3) hours, 144 (±3) hours, 168 (±3) hours, 192 (±3) hours, and 240 (±3) hours. Each patient must complete a minimum evaluable pharmacokinetic sampling up to a 168 hour time point for inclusion in the pharmacokinetic analysis group. Repetitive pharmacokinetic evaluation of rFIXFc was also performed at week 26 using the same sampling time course.

efficacy

A total of 119 subjects were included in the efficacy analysis. The median annual bleeding rate driven by spontaneous bleeding in terms of the 25th percentile and the 75th percentile (interquartile range (IQR)) for team 1, 2 and 3 (ABR), as shown in Figures 3A, 3B and Table 8 and as outlined below:

- Weekly Control Team (Team 1): 2.95 (1.01-4.35)

- Individualized interval control team (team 2): 1.38 (0-3.43)

- Paroxysmal treatment team (team 3): 17.69 (10.77-23.24)

(a) Control dose and interval titration of bleeding events and paroxysmal treatment

All patients were asked to measure the FIX content during the course of the study to estimate the pharmacokinetic parameters of each patient to guide the appropriate dose or dosing interval adjustment to achieve a FIX content above the baseline of 1-3 IU/dl. Standard valley bottom.

In team 1 (fixed weekly interval): all in this team Patients received an initial injection of rFIXFc 50 IU/kg once a week. The rFIXFc dose was adjusted based on monitoring of the trough at weeks 4, 16, 26 and 39. If the pharmacokinetic value indicates that the patient's bottom content is above 3 IU/dl above baseline or higher at the end of the week, the dose is lowered to achieve a target of 1 IU/dl to 3 IU/dl above the baseline. If the pharmacokinetic value indicates that the patient's trough content will be less than 1 IU/dl above baseline at the end of the week, the dose is increased to achieve a target of 1 IU/dl to 3 IU/dl above the baseline. If the target range is not achieved, the plasma sample is manipulated to rule out the presence of the inhibitor. Duplicate trough content was achieved approximately one month after any dose adjustment and repeated monthly until no other adjustments were necessary. Only the dose of rFIXFc was adjusted in this treatment team.

In team 2 (individualized dosing interval), based on patient A dose of 100 IU of rFIXFc per kg was administered intravenously at baseline pharmacokinetic assessment to achieve a target trough of 1 IU/dl to 3 IU/dl above baseline. Monitoring was conducted at weeks 4, 16, 26 and 39 to ensure that the FIX bottom content was maintained. Only the interval of rFIXFc was adjusted in this treatment team.

Unacceptable experience in Team 1 or Team 2 Any patient with blood, two or more spontaneous bleeding for 3 consecutive months, adjusted dose (team 1) or interval (team 2) to achieve FIX trough content above baseline 3IU/dl to 5IU/dl aims. If unacceptable bleeding does not occur at the target trough of rFIXFc above the baseline of 3 IU/dl to 5 IU/dl, it may be re-adjusted at the discretion of the investigator and after reaching agreement with the sponsor. The whole rFIXFc is achieved to achieve a target trough of 1 IU/dl to 3 IU/dl.

In team 3 (paroxysmal treatment), mild bleeding events 20-30 IU/kg, moderate to severe bleeding events treated with 25-50 IU/kg, and severe to life-threatening bleeding events treated with 50-100 IU/kg to achieve 20-30% based on the patient's pharmacokinetic profile, respectively , 25-50% and 50-100% of the bottom FIX content target.

(b) Period of efficacy

In Teams 1 and 2, the efficacy period begins with the date and time of the first dose after completion of the pharmacokinetic sampling period and is recorded in the last cast as reported in the Electronic Case Report Form (eCRF) or Electronic Diary (eDiary). End with dose (used to prevent bleeding events). In team 1 (continuous pharmacokinetics subgroup), the efficacy period was interrupted for a period of repeated repetitive pharmacokinetics, and in teams 1 and 2, the efficacy period was interrupted for all surgical/rehabilitation periods (for major surgery and Both minor surgery). The period of efficacy lasts until the last dose before the repeated pharmacokinetic dose (for the prevention or treatment of bleeding events) or until the last dose before the start of the surgery/rehabilitation period (for the prevention or treatment of bleeding events), followed by pharmacokinetics or surgery / The next dose after the end of the rehabilitation period is restored.

In team 3, the efficacy period begins 1 minute after the last pharmacokinetic sampling time point and ends with the date of the last contact or the date of the last electronic journal entry, whichever is later. The period of efficacy was interrupted 1 minute before the start of the surgery/rehabilitation period and restarted at 00:01 on the day after the end of the surgery/rehabilitation period.

(c) Treatment compliance

Monitored and recorded by the field staff Should be sexual. For administration between visits, the patient self-administers rFIXFc and records the treatment in a hand-held electronic diary that is viewed by the study site staff and clinical monitors during regular phone calls to the patient and at each visit. .

Analysis of patient compliance based on eCRF and electronics Diary information. Compliance analysis includes patient compliance with a given preventive regimen. For weekly control, the analysis included a percentage of the nominal dose taken by each patient in the range of 80% to 125%. Similarly, for individualized interval control, the analysis included the percentage of dose administered to each patient within ± 36 hours of the specified interval. If the calculated compliance rate is at least 80%, consider the patient to be compliant.

(d) Analytical methods

Plasma FIX activity was measured by a single-stage (activated partial clotting hormone time [aPTT]) coagulation assay that was validated for rFIXFc, Bemit and human FIX in human plasma samples. Commercially available aPTT reagents (e.g. Trinity Biotech (Automatic the APTT [based activator and phospholipid mixture of silicon dioxide])), Precision BioLogic (Cryocheck TM removal of plasma factor IX), and as a calibrator in plasma of normal reference (Precision BioLogic) (LLOQ 1 IU/dl) FIX activity in citrate-containing plasma samples was measured on a MDA 180 coagulation instrument having efficacy as specified by the World Health Organization's 3rd or 4th International Standard for Plasma. Accuracy is within 95% to 104% for all three FIX proteins, and intra-assay and inter-assay accuracy is typically within 10%.

Based on the estimates of the negative binomial regression model, the ABR of the two prevention teams was significantly reduced by 83% and 87% compared to paroxysmal treatment (for groups 1, 2 and 3, 3.12, 2.40 and 18.67; P < 0.001, Table 8). The lower ABRs in the control team were consistent across all population-based and disease-related subgroups in the scheduled subgroup analysis (Figures 3 and 2). In addition, in the case of analysis limited to patients in the previous paroxysmal regimen, the ABRs of Teams 1 and 2 were reduced by 83% and 89%, respectively, consistent with the primary analysis. For the prevention and treatment period during the study, the proportion of patients without any bleeding event was 23.0% for team 1 and 42.3% for team 2. In addition, analysis of bleeding events by type and location showed a lower ABR in both control groups (Table 8). Subjects in team 1 had a weekly dose of rFIXFc (IQR) of 40.5 IU/kg during the last 3 months of the study. The dosing interval of approximately half of the study population in the individualized interval control team (team 2) during the last 6 months of the study 14th. A summary of the median administration of Team 1 and Team 2 is shown in Tables 9A and 9B.

^a based on research 6 months of subjects

IQR = 25th percentile and 75th percentile.

IQR, quartile range

^† Allow pharmacokinetic-driven administration (team 1) or interval (team 2) to achieve a bottom content of 1% to 3% above baseline. If the patient has two breakthrough spontaneous bleeding events during the 3-month rolling period, the dose (team 1) or interval (team 2) can be adjusted to provide additional protection.

^{I am} in the study Among patients who are 9 months old

^§ In research Among patients who are 6 months old

ABR indicates annual bleeding rate, CI indicates confidence interval, IQR indicates quartile range (25th percentile and 75th percentile), rFIXFc indicates recombinant Factor IX Fc fusion protein, and SD indicates standard deviation.

^* Excellent: Sudden pain relief and/or bleeding signs improvement within approximately 8 hours after initial injection; Good: Clear pain relief and/or bleeding signs improvement within approximately 8 hours after injection, but may require 24 to 48 hours More than one injection is then administered to achieve a complete resolution; medium: there may be a possible or slightly beneficial effect 8 hours after the initial injection and more than one injection is required; no response: no improvement or worsening of the condition within about 8 hours after the initial injection.

^** Excellent: The bleeding event responds to rFIXFc less than or equal to the usual number of injections or less than or equal to the usual dose of rFIXFc, or the breakthrough bleeding rate during the control period is less than or equal to the commonly observed breakthrough bleeding rate. Effective: Most bleeding events respond to the same number of injections and doses, but some require more injections or higher doses, or there is a small increase in breakthrough bleeding rates. Partially effective: bleeding events most often require more injections or higher doses than expected, or sufficient breakthroughs during prevention and treatment to require more frequent injections and/or higher doses. Invalid: Other procedures are not required for routine procedures that do not control hemostasis or hemostasis control.

Control of bleeding: A total of 636 bleeding events (team 1 = 167, team 2 = 67, team 3 = 402) were treated in 89 subjects, with more than 90% (90.4%) bleeding events by single Injection of rFIXFc was controlled and 97.3% by Two injections were controlled. Regardless of the type, compliance status, or location of the bleeding event, the number of median injections required to resolve the bleeding event was 1.0, which excluded two internal bleeding events that were required to be resolved by two injections in Team 2. The median dose for each injection was 46.1 IU/kg and the median dose for the bleeding event was 47.0 IU/kg. For a 61 (9.6%) bleeding event requiring >1 injection, the median interval between the first injection and the second injection was 45 hours. Overall, subjects in groups 1, 2, and 3 were rated excellent or good for the response to rFIXFc treatment for 76.4%, 77.0%, and 85.4% of the injections, respectively. In teams 1, 2, and 3, for 98.8%, 99.2%, and 97.9%, respectively, the physician's overall assessment of the subject's response to his rFIXFc regimen was rated as superior or effective. Bleeding events are summarized in Table 10.

When based on each subject and for treatment after symptom onset Subjects with an event of more than 8 hours were similar when examined between teams.

The dose interval for subjects in team 2 increased from the initial 10 days to 53.8%. Subjects reached a median dose interval during the last 3 months of the study. 14th.

Perioperative management: The end of team 4 includes the investigator/surgeon's assessment of the subject's response to surgery with rFIXFc: the number of injections and doses required to maintain hemostasis during the surgical period; during and after surgery Estimated blood loss; and the number of blood transfusions required for surgery. Overall, 14 major surgeries were performed in 12 subjects, including knee arthroscopy (n=4) and ankle arthroscopy (n=1), knee replacement (n=4), and other procedures (n =5). Hemostasis was rated as excellent (13/14) or good (1/14) by the investigator/surgeon. The median estimated blood loss was 65.5 mL (range: 0.0-300.0 mL) during surgery and 0.0 mL after surgery (range: 0.0-500 mL). No blood transfusion was required during the procedure, but two subjects received blood transfusions during the postoperative period. In 85.7% of the operations, a single injection of rFIXFc was required at each injection of a median dose of 90.9 IU/kg to maintain hemostasis during surgery. One day before the surgery, 1-2 injections were administered and 2-3 injections were administered during the 3 days after surgery. Median rFIXFc consumption (summarized for all injections during the reference period) on days of surgery, 1-3 days after surgery, and days 4-14 after surgery, were 146.1 IU/kg, 168.2 IU/kg, and 277.1, respectively. IU/kg. Overall, 10 (83.3%) of the 12 subjects reported 1 adverse event (AE) and reported 3 subjects with 6 severe AEs, all of which were resolved and judged to be unrelated to rFIX treatment. Therapist assessed the hemostasis efficacy of rFIXFc in 100% surgery as excellent or good.

One injection is enough to solve 90.4% of the bleeding in Team 1. A median dose of 46 IU/kg per injection. Overall, 82.0% of rFIXFc injections were assessed by the subjects to produce excellent or good responses. For 98.8% of the subjects, the physician's overall assessment of the subject's response to rFIXFc was rated as superior or effective.

The weekly rFIXFc dose of subjects in team 1 decreased from the initial 50 IU/kg to a median of 45 IU/kg during the course of the study. The initial dosing interval of subjects in team 2 increased to 12.5 days through the study period, with 12 subjects (46%) achieving dose intervals in the last 6 months. On the 14th, 14 subjects (53.8%) reached the dose interval in the last 3 months of the study. 14th.

For 14 major surgeries in 12 subjects 100%, the hemostasis in team 4 is rated as excellent or good by the investigator or surgeon. Types of major surgery include treatment of dental abscesses and hair follicles, as well as knee arthroplasty or knee replacement (n=6) and interception (n=2). A single injection of rFIXFc was sufficient to maintain hemostasis during the 85.7% major surgery and the median average dose for each of the 14 procedures was 91 IU/kg.

Overview of hemostasis response to administration during and after surgery In Table 11.

PK

For FIX active pharmacokinetic analysis, baseline (endogenous FIX activity) is pre-defined as the lowest observed FIX activity at the time of screening, before administration, after administration, or according to the patient's historical clinical record. Baseline FIX activity was set to zero for patients with a minimum observed FIX activity of less than 1%; for patients with a minimum observed FIX activity between 1 IU/dl and 2 IU/dl, baseline FIX activity was set to the actual observed FIX value. The residual drug is attenuated following a first order decay, wherein the rate of decay is determined individually. An overview of FIX activity corrected by baseline and residual drug over time was analyzed using a user-defined and validated dual compartment model. The user specification code automates the calculation of other secondary pharmacokinetic parameters (eg, 1% and 3% time), which are not included in the secondary parameter list of the WinNonlin library model (PHOENIX ^® WinNonlin 6.2.1.51; Pharsight). Therefore, the need to manually process data beyond the primary analysis is eliminated and the risk of introducing human error is minimized.

rFIXFc geometric mean terminal half-life of about 82 hours, the ratio of 96- = BENEFIX ^® hour of sample geometric mean terminal half-life (about 34 hours) 2.4-fold (i.e. 2.43-fold) (p <0.001) long. At 48 hours of sampling, a half-life increase of 4.83 times was observed. Compared with rFIX, rFIXFc has a mean residence time (MRT) of 2.39 times longer, resulting in a 2.21 fold and a 2.04 fold (P < 0.001) increase in time to reach 1 IU/dL and 3 IU/dL, respectively. 21 subjects were compared at baseline with rFIXFc at week 26 and showed no statistical difference in any PK parameters. Table 12 shows comparative PK data for rFIXFc versus rFIX from 22 subjects in a continuous PK subgroup of Team 1 with a compartmental model for single stage coagulation analysis.

As shown in Table 12, increment between rFIX and rFIXFc The recovery was similar (P=0.713). When the 96-hour sampling time course was used, there was a 2.43 fold increase in terminal FIX half-life after rFIXFc compared to rFIX (82.1 vs. 33.8, respectively; P < 0.001; see Table 12 and Figure 4). Using a conventional 48-hour sampling time period that has been used to show the generally accepted half-life (about 18 hours) of rFIX, rFIXFc showed a 4.84-fold increase in half-life compared to rFIX. Compared with rFIX, the mean residence time of rFIXFc (MRT: 41.2 vs. 98.6 hours; P < 0.001) was 2.39 times longer, resulting in a 2.21 fold increase in time to reach 1 IU/dL above baseline (5.1 days vs. 11.2 days; P < 0.001). The time for rFIXFc to reach 1 IU/dL (1%) FIX and the time to reach 3 IU/dL (3%) IU/dL were 11.2 days and 5.8 days, respectively (see Table 12 and Figure 4). The in vivo rFIXFc PK parameter was comparable between baseline and week 26, indicating that the PK profile of rFIXFc was stable after repeated administration for 26 weeks.

All subjects underwent initial PK assessment to characterize PK of rFIXFc in a representative population of subjects with hemophilia B.

50IU / kg single dose of BENEFIX ^®, followed after 50IU / kg single dose of rFIXFc, more subjects in the Control group team (team group 1) in one subset at baseline in the weekly Extensive PK sampling. Blood samples of BENEFIX ^® were obtained over a period of 96 hours. Blood samples of rFIXFc were then acquired over a period of 240 hours. The PK assessment of rFIXFc was repeated at 26 weeks.

Select 100-IU/kg agent based on PK results of Phase 1/2 study Quantity, these results show that this dose raises the FIX content to about 100% of normal (Shapiro et al. 2011). In the 1/2 phase study with 100 IU/kg rFIXFc (n=5), the time to reach a FIX content of 1% above baseline was about 11 days, and was in the range of 9 to 14 days. Based on this information, Team 2 was designed to test whether a fixed dose of 100 IU/kg could provide protection against bleeding for more than a week.

rFIXFc results in a lower median ABR in the weekly control team The middle is 2.95, which is 1.38 in the individualized interval prevention team. In contrast, the ABR of the paroxysmal treatment team was 17.69.

In the individualized interval prevention team, at the end of the study 6 The median dosing interval was 14 days during the month.

The single-stage clotting rFIXFc by measuring the terminal half-life analysis (activity) of about 82 hours (82.1 hours) and BENEFIX ^® is about 34 hours (33.8 hours).

Overall, more than 90% of bleeding events are by a single bet Shooting is controlled. In 100% of the operations (14 major surgeries in 12 subjects), the hemostatic efficacy of rFIXFc for perioperative management was rated as excellent or good by the treating physician.

rFIXFc weekly prevention team and individualized interval team The value ABR is 2.95 and 1.38 respectively.

More than 90% of bleeding events were obtained with a single rFIXFc injection solve.

About 34 hours of measurement of the BENEFIX ^® B-LONG terminal half-life is longer than in BENEFIX ^® package insert reported the terminal half-life (about 18 hours) and 48 hours using the sampling duration, followed EMA FIX guidelines on the assessment of PK The terminal half-life (13.7 hours to 19.3 hours) reported in many studies (Ewenstein, 2002; Kisker et al., 2003; Negrier et al., 2011). However, on the PK study public until 72 hours after the administration BENEFIX ^® sampling, also reported a longer terminal half-life is 21.3 to 33.4 hours (Ragni et al., 2002, Lambert et al., 2007, Chang et al., 2007 And Martinowitz et al., 2012).

To determine if the difference in terminal half-life of BENEFIX ^® was due to the longer 96-hour PK sampling timeframe used in this study, the BENEFIX ^® PK data was also analyzed using data up to 48 hours after dosing. This analysis produced a significantly shortened BENEFIX ^® terminal half-life (approximately 17 hours) consistent with previous reports using a 48-hour sampling duration. The BENEFIX Summary Comparison of the data presented in the Table ^® 12B.

In the B LONG-study, performed head to head comparison between rFIXFc and BENEFIX ^®, and in phase 1/2 study, historical data reported in the half-life of rFIXFc with BENEFIX ^® product insert (2009) are compared. The rFIXFC B-LONG study of over BENEFIX ^® PK modified for more reliable and accurate measurements.

safety

The tolerance to rFIXFc is good and has a significant improvement in PK relative to current care standard rFIX. One severe AE (obstructive uropathy) was assessed as far as possible in connection with the treatment; the event was resolved and the subject continued to be in the study. Good control of bleeding and low ABR seen during weekly to biweekly (every 2 weeks) indicates that this therapy can substantially improve the management of acute bleeding and significantly prolong the preventive regimen in hemophilia B. This may improve subject compliance and outcome.

To detect inhibitors (neutralizing antibodies), a Nijmegen-modified Bethesda assay to detect neutralizing antibodies was performed at each visit during screening, baseline, and study treatment to monitor the production of inhibitors. After the first dose of rFIXFc, an inhibitor test was performed at the bottom of each of the scheduled clinical visits in teams 1 and 2, where the bottom was defined as the point after the longest interval between predetermined doses. Inhibitor testing was performed in team 3 after at least 72 hours (3 days) of elution. For new patients entering team 4, an inhibitor test was performed at screening and then a total of at least 4 EDs were achieved with rFIXFc within 4 weeks prior to the scheduled surgery. The formation inhibitor system is defined as confirming the neutralizing antibody value in the second separate sample within 2-4 weeks. 0.6 Bethesda units (BU) / ml. A confidence interval for the incidence of inhibitors was evaluated using the Klopp-Pearson binomial proportional method. The risk of inhibitors acceptable in clinical trials of previously treated FIX patients allowed one of 50 patients to undergo an inhibitor at the time of the study, with each patient requiring at least two effective inhibitor tests over 50 to 75 EDs.

In order to detect non-neutralizing antibodies (rFIXFc binding antibodies), Non-neutralizing antibodies (NNA) bound to rFIXFc were monitored at the same time points as for the inhibitors. A bridge analysis format was used to detect antibodies of all possible classes, with electrochemimetric readings being read on the MSD instrument. The sample is positive if the signal is above the statistically obtained segmentation point and confirmed by inhibition of excess rFIXFc product. The positive sample is further characterized for binding to rFIX or Fc. Testing of inhibitor positive control samples showed that this analysis was approximately 100 times more sensitive than the Nijmegen-modified Bethesda assay.

The study was designed to include 50 ED 50 subjects were tested for the risk of an inhibitor. The US Food and Drug Administration's guidance to fully demonstrate the risk of acceptable inhibitors in clinical trials of previously treated FIX subjects allows one of 50 subjects to undergo an inhibitor, Each subject needs 50 studies treated ED. Under the hypothesis that the occurrence of inhibitors in clinical studies can be fully modeled using the binomial distribution, if one inhibitor is observed, 50 EDs would allow for a 2-sided 95% confidence interval for true inhibitor incidence (0.05% - 10.65%) using the exact Clopper-Pearson method. Including 55 ED 50 subjects and 94 EDs No death, allergic reaction, thrombotic event, or inhibitor production was reported in any of the study subjects of the 25 patients. No inhibitors (neutralizing antibodies) were detected in any of the subjects in the study. Low positive results for non-neutralizing antibodies (NNA) were found in 3 subjects at baseline, which did not affect the PK of rFIXFc and all 3 subjects recovered NNA negative status during the study. One subject had a border-negative result during the study and had a border-positive result at the end of the study. rFIXFc clearance was not affected and NNA was not associated with any increased bleeding frequency and/or increased rFIXFc consumption. The incidence of AE was similar across all treatment teams, with 45 participants (71.4%) in Team 1 and 23 subjects (79.3%) in Team 2 and 20 participants in Team 3 (74.1%) of AE 1. Most AE lines are judged by the investigator to be unrelated to treatment or less likely to be associated with treatment. AEs in 10 of 119 subjects (8.4%) of the combined team 1, 2, and 3 were reported to be likely to be treatment-related or treatment-related. No discernible trend of coagulation activation markers (F1+2, D-dimer, thrombin-antithrombin complex) was detected in the continuous PK subgroup, and was not detected in any of the teams. A discernible trend in total IgG and subclass content.

Long-lasting rFIXFc is effectively maintained in subjects with hemophilia B Perioperative hemostasis. All the big hands carried out by surgeons and researchers A high assessment of perioperative hemostasis during surgery indicates that blood loss is comparable to the expected blood loss for subjects without hemophilia.

For both weekly control and longer interval duration (eg, every 1 - 2 weeks) and/or higher trough content, the results disclosed herein show that rFIXFc provides effective control of acute bleeding events and displays Effective prevention. It is worth noting that about 50% of the subjects in the individualized control group (team 2) showed the dosing interval. 2 weeks. Compared to rFIX, rFIXFc showed a substantial improvement in the PK profile, allowing the prophylactic regimen to use a longer dosing interval that would be less burdensome for the subject. The PK profile of rFIXFc is stable over time. These results show that rFIXFc is the first long-lasting recombinant Factor IX therapy in its class, which provides treatment for less frequent injections and still provides long-lasting defense against bleeding, thereby promoting increased adherence to control and type B The results in hemophilia subjects improved.

To ensure consistency in laboratory analysis, a center will be The laboratory is used for each type of analysis. Analysis of clinical safety samples and central sample management were performed at LabCorp (Cranford, NJ, USA). FIX activity and inhibitor assays were performed at LabCorp (Englewood, CO, USA). The genotype of the samples was determined at the Hemostasis Lab, Puget Sound Blood Center (Seattle, WA, USA). FIX antigen and non-neutralizing antibody (NNA) samples were analyzed by ICON Development Solutions (Whitesboro, NY, USA), and rFIXFc concentrated samples were analyzed by Chimera Biotech GmbH (Dortmund, Germany).

Early termination when all of the following predetermined criteria are met the study:

1) Thirteen patients in the continuous pharmacokinetic subgroup of team 1 completed pharmacokinetic sampling to fully estimate the terminal half-life of the shellfish at baseline and the terminal half-life of rFIXFc at baseline and week 26.

2) 53 patients from any treatment team completed at least 50 rFIXFc EDs.

3) Twenty patients from team 2 and 16 patients from team 3 completed at least 26 (±1) weeks of study.

4) 73 patients from any treatment team completed at least 50 rFIXFc ED and were tested for inhibitors; or 53 patients completed at least 50 EDs and were tested for inhibitors, as confirmed by retests, up to 1 The patient has a positive inhibitor; or 34 patients complete at least 50 ED and are subjected to an inhibitor test, wherein no patient has a positive inhibitor as confirmed by retesting.

5) Perform about 10 major surgeries in at least 5 patients and complete post-operative follow-up.

When all of these criteria have been met, all ongoing patients are required to return to the clinic to complete the study assessment.

Impact on quality of life

The quality of life was measured using a hemophilia specific quality of life measuring instrument, HAEM-A-QoL. HAEM-A-QoL was performed in adults (18 years and older) in the preventive treatment team. The changes from the baseline at week 26 in the control team that were combined according to the pre-study protocol are summarized in Table 13.

During the last 6 months of the study, the median dosing interval in the individualized interval control team was 14 days.

Control of bleeding: More than 90% (90.4%) of bleeding events were controlled by a single injection of rFIXFc.

Perioperative management: Therapist assessed the hemostasis efficacy of rFIXFc in 100% surgery as excellent or good.

Ten (8.4%) of the 119 subjects treated with conventional control or paroxysmal (on-demand) therapy reported adverse drug response (ADR). Adverse drug reactions are considered to be adverse events that are assessed by the investigator as being associated with rFIXFc therapy or that may be associated with rFIXFc therapy. Unfavorable drug reaction These are described in Tables 14A and 14B.

The incidence of adverse events is similar across all treatment teams. Among them, 45 patients (71.4%) in team 1 and 23 patients (79.3%) in team 2 and 20 patients (74.1%) in team 3 had at least one adverse event. Most are judged by the investigator to be unrelated to treatment or impossible to treat. Adverse events that were judged to be treatment-related or possibly treatment-related were reported in 10 (8.4%) of the 119 patients in pooled teams 1, 2, and 3. A serious adverse event that may be considered by the investigator as a possible treatment is a urinary system obstructive clot, which is solved by hydration and the patient continues to study treatment. There were no reports of allergies, allergies or thrombotic events in the study and there was no death. There are no clinically significant changes in the levels of coagulation activation markers (F1+2, D-dimer, thrombin-antithrombin complex) or total IgG and subclasses.

No subjects withdrew from the study due to adverse drug reactions. In the study, no inhibitors were detected and no allergic events were reported.

The tolerance to rFIXFc was good with no signs of new antigenicity and the adverse events were consistent with the adverse events expected in the hemophilia population. It is worth noting that the high compliance rate (96.6%) and the corresponding high study completion rate (93.5%) in this study further support the tolerability of rFIXFc. The risk of inhibitors in the natural hemophilia B population has been reported to be as high as 3%. See Tandra A and Shapiro AD (2010), Lee CA, Berntorp E, Hoots WK, Textbook of hemophilia, 2nd edition Hoboken, NJ: Wiley-Blackwell; and DiMichele D. (2007) Br J Haematol 138 :305-15 . Patients typically develop inhibitors within 50 EDs prior to exogenous FIX replacement therapy (see Dimichele D. (2002) Haemophilia 8 : 280-287), which supports the end of the study guidelines. B-LONG includes a total of 5144 EDs (117.1 patient exposure years), of which 55 patients have 50 EDs and no inhibitors were detected in any of the patients. In this study, low titer NNA was observed in a small proportion of patients (3%) at screening and baseline and became negative in three of the four cases during rFIXFc treatment. The low potency NNA observed in this study did not have a discernible effect on rFIXFc pharmacokinetics or bleeding rate.

Example 5. Population pharmacokinetics of rFIXFc clinical significance in routine control, bleeding control, and perioperative management of subjects with hemophilia B

BACKGROUND: Clinical administration of Factor IX (FIX) in the treatment of hemophilia B is well established based on empirical practice and clinical outcomes. Since the pharmacokinetics (PK) of FIX activity is an alternative efficacy marker, we used population PK (popPK) modeling and simulation to evaluate the dosing regimen of the long-acting recombinant FIX Fc fusion protein (rFIXFc). Best described by a 3-compartment model rFIXFc PK from 135 single doses and 21 repeated dose profiles in 12-year-old subjects (body weight (BW): 45-186.7 kg), this model shows moderate inter-subject variability in clearance (CL) (IIV) Moderate inter-subject variability of 17.7% and central compartment volume (V1) was 21.7%. The proportional residual error of 10.6% is similar to the variability of single-stage coagulation analysis of FIX activity. The only covariate showing a weak association with rFIXFc PK is BW, which accounts for about 3% of the IIV of CL and V1, indicating that BW-independent rFIXFc flat administration can be feasible for treating adult hemophilia B subjects.

Purpose: To simulate the routine use in the hemophilia B population BW-based and flat dosing regimen for control, bleeding control, and perioperative management.

Methods: A validated 3-compartment popPK model including inter-temporal variability and BW as a covariate of CL and V1 was used for dosing simulation. For the BW-based dosing regimen, a PK profile of 1000 subjects was simulated, where the BW profile represents a phase 3 study. The BW distribution is simulated using the power function Z=BW-0.5. The resulting BW (1000 values) distribution has a median value of 74.9 kg and 38.9 kg to 172.6 kg, which is similar to our study (median 73.3 kg; minimum and maximum, 45 kg and 186.7 kg). For fixed dosing regimens, based on low ( 10th percentile), typical (10th - 90th percentile) and high ( The 90th percentile) BW is layered for three populations (n each equals 1000). The variability of the exposure parameters, the percentage of populations that maintained the target Cmax and trough, and the deviations of the median exposure parameters in the extreme BW group were compared to a BW-based and flat dosing regimen. To mimic the steady state in the control regimen, six doses were used for all dosing regimens (once a week, every 10 days or every 14 days), with each dosing interval being assigned to one situation. To simulate the PK profile after paroxysmal treatment, a single dose was applied.

RESULTS: Consistent with observations from Phase 3 studies, a weekly 50 IU/kg or 100 IU/kg per day 10 IU/popPP simulation predicted that in most of the population, FIX peak activity was within the physiological range (Cmax < 150%). And the bottom 1%. All simulations predicted that most of the population would maintain a trough activity at or above 1% (Table 15).

A population simulation showing steady-state FIX activity versus time The figure is shown in Figure 13. Table 16 shows steady state FIX activity predicted over the course of 14 days for the following two dosing regimens: 50 IU/kg per week and 100 IU/kg per 14 days.

In addition, BW-based and flat-administered administration yielded a PK profile comparable to comparable exposure parameters, such as weekly predicted 50 IU/kg and 4000 IU predicted median (5th, 95th percentile) Cmax of 52.6 (32.1, respectively). 89.3) IU/dL and 56.1 (36.2, 90.9) IU/dL. Both dosing regimens predict >95% in the population to maintain Cmax < 150% and bottom 1% (Figure 14). However, BW-based and flat drug delivery is shown at the extreme ( 10th or The 90th percentile) BW population has a differential effect on exposure parameters. This suggests that BW-independent flat administration is feasible for patients 12 years of age and older.

Use the popPK model to simulate the administration of paroxysmal therapy case. The model predicts control of bleeding events. A single dose of 50 IU/kg or 100 IU/kg of rFIXFc is sufficient to maintain plasma FIX peak activity at 40 IU as recommended by the World Federation of Hemophilia (WFH) guidelines. dL to 80 IU/dL (Table 17).

Analysis of 12 major surgeries and 2 minor surgeries found The FIX activity measured during the surgical period was essentially consistent with the prediction by popPK based on the pre-operative baseline PK of the subject, indicating no substantial factor consumption during these procedures. A representative graph of the observed and predicted perioperative FIX activity is shown in FIG. Simulated FIX activity and observed FIX activity within the first 21 days after the first rFIXFc surgical dose (n=14; 12 major surgeries, 2 minor surgeries). There was a good correlation between the observed FIX activity data and the FIX activity data predicted by the PK model (relative prediction error [95% CI], 0.332% [-2.08%, 1.42%]).

Conclusion: popPK provides a way to evaluate potential dosing regimens Robust and effective means. The results of Phase 3 studies were confirmed by prediction of rFIXFc by popPK simulation. A similar PK profile was achieved for the simulation of BW-based flat administration of rFIXFc. Considering the broad therapeutic range of factor replacement therapy, flat administration of rFIXFc and rFIX products can be one A method of permitting other clinical studies that is potentially feasible in adult hemophilia B subjects. In addition, under the population PK model, it is feasible to generate general dosing instructions to achieve the target FIX content recommended for perioperative management of patients with hemophilia B.

Example 6. Population pharmacokinetic analysis of long-acting recombinant Factor IX-Fc fusion protein (rFIXFc) in subjects with severe hemophilia B

BACKGROUND: A population pharmacokinetic (PK) model is generated to understand the source of dose requirement (covariation) variability and, if necessary, to help individualize the dosing regimen. The history of administration and subject-specific data were used to obtain an understanding of the drug configuration to identify specific demographic data and/or clinical factors that may be predictors of PK parameters. Characterization of long-acting FIX-Fc (rFIXFc) in severe hemophilia B (plasma FIX activity) 2 IU/dL) Population PK (popPK) in the subject, an estimated population PK parametric model of rFIXFc can be established. This model can help physicians who wish to customize the administration of individual subjects with a rare PK sample.

METHODS: Male Severe Type B hemophilia subjects from the 1/2a phase (n=12) and Phase 3 studies (B-LONG, n=123) were included. Subjects ranged in age from 12 to 76 years and ranged in weight from 45 kg to 186 kg. The modeled data set included 135 baseline PK profiles at week 1 and 21 replicate PK profiles at week 26, with a total of 1400 measurements of FIX activity. The final population PK model was validated using 1027 trough/peak FIX activity records from 119 subjects.

In the popPK assay, plasma FIX activity was measured by single-stage (activated partial clotting hormone time) coagulation analysis. Corrected FIX activity using the following formula: corrected FIX activity = measured FIX activity - baseline - residual decay.

Baseline FIX activity is defined as the minimum activity level (LLACT) at the time of screening, before administration, after administration, or from historical clinical records. When the baseline is equal to 0, LLACT is less than 1% (lower limit of quantitation). When the baseline FIX activity is equal to LLACT, LLACT is greater than or equal to 1% and less than or equal to 2%.

The pre-study residual decay was performed using the terminal half-life obtained from non-compartmental analysis of individual data by the following equation: residual decay = (pre-dose-baseline) x e ^{- decay rate x time} .

To generate the popPK model, version 1.0 NONMEM VII (ICON Development Solutions, Ellicott City, Maryland) was used. The modeling and identification steps are presented in Table 20 below.

One-stage conditional estimation (FOCEI) was performed using an interaction method to estimate the popPK parameters. The residual error is modeled in the form of combined proportional error and additive error. A covariate modelling was performed with progressively forward addition (p < 0.005) and backward elimination (p < 0.001). The potential covariates evaluated in this analysis included body weight (BW), age, ethnicity, blood type, human immunodeficiency virus status, hepatitis C virus status, hematocrit, IgG ₁ and albumin concentrations, and FIX genotype.

Model identification includes shoe re-extraction, visual predictive inspection (VPC), and verification with valley/peak records. The average relative prediction error (indicator of accuracy) is calculated as:

RESULTS: The rFIXFc configuration was made up of the three-compartment base model. Good description (Figure 5). The model was further improved by including a random variation of CL and V1 in subjects in different situations (i.e., inter-temporal variability IOV) (Fig. 6). IOV is less than inter-subject variability (IIV), indicating that individual PKs predict individual PK more accurately than mean popPK.

Found that the weight is the effective covariate of the rFIXFc configuration (Figure 7), but the impact of BW is limited. For example, the BW indices for CL and V1 were 0.436 and 0.396, respectively, and inclusion of BW only reduced inter-subject variability (IIV) for both CL and V1 by 3.4% and 2.5%, respectively. Other assessment covariates, including age, race, blood type, or genotype, are not valid covariates in this model.

The final popPK model is summarized in Table 21 below.

Clear clearance, central compartment for typical 73 kg subjects The predicted popPK values for the product and the steady-state volume are 2.39dL/h, 71.4dL and 198dL, respectively. The goodness-of-fit plot shows the rigorous simulation of FIX activity data by predictive popPK data generated by the model (Fig. 8).

Verify the knot of the popPK model using observed FIX activity data fruit. The median and 80% intervals of the observed and predicted FIX activity time profiles almost overlap, indicating that the final model was able to reproduce the concentration trend and variability of the observed FIX activity data on a time scale (Figure 9). A strong correlation between observed FIX activity in the bottom/peak dataset and predicted FIX activity indicates that the final popPK model is predictive (Figure 10).

Finally, the total relative prediction error is -3.23%, 95% of which The confidence interval is -5.27% to -1.23%. The post-equivalent estimates for this popPK analysis are very similar to the results of the conventional PK analysis shown in Table 22 below.

Conclusion: The three-compartment popPK model predicts moderate testing Inter-invariant (IIV) rFIXFc configuration. The individual PK parameters generated by the three-compartment popPK model are similar to the individual PK parameters generated by the two-compartment PK analysis, indicating that the contribution of the third compartment is limited. For a typical 73 kg subject, the popPK model predicted a clearance rate of 2.39 dL/h; the central compartment volume was 71.4 dL; and the steady-state volume was 198 dL. The only valid covariate evaluated in the popPK model is BW, but its effect on rFIXFc PK variability is limited.

The final popPK model can be used to simulate routine management, control, and prevention of bleeding events, as well as perioperative management of dosing schedules and intervals. This model can help physicians who wish to customize the administration of individual subjects with a rare PK sample.

Example 7. Pediatric research Research design

Description to evaluate rFIXFc control and prevention of previously treated severe hemophilia B (endogenous FIX activity) 2IU/dL [2%]) Phase 3 open-label multicenter pediatric study of the safety, PK, and efficacy of bleeding in children (<12 years).

Approximately 26 children with hemophilia B were recruited. This pediatric pre-treatment subject (PTP) study eligibility guidelines require that the child's previous recorded exposure date (ED) for the FIX product has been 50, weight when agreeing 13kg, and currently does not have an inhibitor of FIX products or a history of inhibitors without FIX products. The treatment regimen is for control (see the study design shown in Figure 11). PK and rFIXFc were subjected to PK analysis in the participants prior to initiation of prophylactic treatment with rFIXFc.

Primary and secondary outcome measures will include the frequency of inhibitor production (for primary outcome measures) and the number of annual bleeding events and/or responses to treatment of bleeding events with rFIXFc (for secondary outcome measures).

Will also evaluate rFIXFc in previously untreated subjects (PUP) For safety and efficacy, previously untreated subjects were defined by the European Medicines Agency as subjects who were not previously exposed to any factor product. The results of the PTP and PUP studies in children will provide further insight into the clinical safety and hemostasis parameters of rFIXFc in children with hemophilia.

Interim analysis

Control with Factor IX (FIX) is the best treatment for patients with hemophilia B; however, due to the short half-life of currently available FIX products, frequent injections may be required to prevent bleeding events. To extend the half-life and reduce the frequency of injection, the development of covalent attachment to immunoglobulin G1 A durable recombinant FIX Fc fusion protein (rFIXFc) consisting of one rFIX molecule of the Fc domain of (IgG1). In a phase 3 study in adults and adolescents, the half-life of rFIXFc increased by 2.43 times and the clearance (CL) decreased by 50% compared to FIX (BeneFIX®) (J Thromb Haemost. 2013; 11 [2]: 241). . The Children's B-LONG study (NCT01440946) was designed to evaluate the pharmacokinetics (PK), safety and efficacy of rFIXFc control in previously treated pediatric subjects with type B hemophilia. The goal of this planned interim analysis was to determine the PK parameters of subjects recruited by rFIXFc in childhood B-LONG and compare these parameters to their pre-study FIX PK parameters.

METHODS: This multicenter open-label phase 3 study is currently recruiting patients with severe hemophilia B ( 2 IU/dL endogenous FIX), previously treated subjects with an exposure date (ED) for FIX products of at least 50 and no FIX inhibitors <12 years old. Subjects were stratified into two age groups (<6 years and 6 years old to <12 years old). A weekly prophylactic regimen of 50-60 IU/kg rFIXFc was administered to all subjects, with subsequent doses and intervals adjusted based on PK data and bleeding frequency. Subjects will continue treatment until they reach 50 EDs. The primary endpoint is the formation of inhibitors. Continuous PK analysis was performed to compare the PK parameters of rFIXFc with the PK parameters of the pre-study FIX product. PK sampling of FIX before the study occurred at baseline before the first dose of FIX (50 IU/kg) and at 5 other time points up to 48 hours. PK sampling of rFIXFc occurred before the first dose of 50 IU/kg rFIXFc and after the first dose up to 7 other time points of 168 hours; 96 hours before both the FIX and the first dose of rFIXFc before the first dose study The elution period. Using a single-stage coagulation analysis with respect to the measurement of plasma FIX activity in plasma FIX commercially available standards of calibration, and data analysis software used PK PHOENIX ^TM WinNonlin 6.2.1.51, by non-compartmental analysis (NCA) was analyzed over time of FIX activity Overview. The information with the deadline of April 23, 2013 is used to report the PK data in the mid-term analysis.

RESULTS: During the interim analysis, 24 subjects were recruited and It has received at least one dose of pre-study FIX and/or rFIXFc. Of the 18 subjects evaluable in the PK profile, 15 had a complete PK profile of pre-study FIX (BENEFIX®, HAEMOSOLVEX® or ALPHANINE®) and rFIXFc. A comparison of the PK parameters of rFIXFc versus FIX for the two age groups is presented in Table 23. Compared to FIX products, the half-life of rFIXFc is extended by more than 3 fold and CL is reduced by more than 60%.

Conclusion: rFIXFc compared to currently available FIX products It has an extended half-life and a reduced CL in pediatric subjects, similar to previous observations in adults and adolescents. The final analysis of the B-LONG study in children will provide further PK information and evaluate the safety and efficacy of rFIXFc in children.

Example 8. Using a population pharmacokinetic model of rFIXFc to simulate or estimate treatment information for individualized and median patients

As discussed in Examples 5 and 6, a model of estimated population PK parameters for rFIXFc has been established that can assist physicians and other healthcare practitioners who wish to customize dosing for individual subjects, such as PK samples that are sparse. Alternatively, the model can be used to determine dosing based on PK data (median PK) for the entire population.

Thus, Bayesian estimates (or similar machine learning algorithms) can be used to select, for example, pharmacokinetic (PK) and dosing regimens based on the population pharmacokinetic (popPK) model described in Example 6 above (eg, Table 21). Individualized patient treatment. In this way, alternative preventive dosing regimens and optimized dosing regimens that have not previously been studied in the B-LONG trial for perioperative management can be determined. Alternatively, the selected dosing regimen is based on population PK (median PK) rather than individualized selection.

In some embodiments, the rFIXFc popPK model of Example 6 (e.g., Table 21) was used without Bayesian or similar machine learning algorithms.

In some embodiments of this aspect of the invention, the method is performed on a computer based system, such as a server, desktop, laptop, tablet, handheld device, or smart phone. In some embodiments, the computer based system is a computer application. The computer based system includes storage media for the rFIXFc popPK model discussed in Example 6, such as the parameters of Table 21. In some embodiments, the storage medium may also contain a Bayesian estimation program, such as NONMEM or Phoenix NLME. For example, Example 6; Kiang et al, Clin . Pharmacokinet 51 : 515-525 (2012).

In some embodiments, the system includes two or more Computer based system. In some embodiments, the user can enter information into the first computer-based system in communication with the second computer-based system, and the second computer-based system performs the calculation and communicates the output information to the first A computer based system. This output information may include recommendations for individualized or non-individualized dosing regimens.

In some embodiments, the user enters information into the system And the system calculates and outputs one or more PK or dosing regimens. In some embodiments, the system uses the received information to calculate and output the individualized or median PK information. In some embodiments, the system calculates individualized dosing or interval information.

Information that can be input by the user and received by the system includes the patient Information and information on the outcome of the treatment. Based on the type and value of the information received, the computer based system calculates the output information based on the rFIXFc popPK model on the storage medium and the machine learning algorithm selected as appropriate.

Patient information includes, for example, age, weight, and diagnostic (baseline) FIX content, PK measurement value, PK sampling time, administration history (if PK sample is taken from multiple doses), actual dose, degree of FIX activity, and the like.

The information to be treated includes the desired PK or desired solution. For example, the degree of plasma FIX activity is increased after the dose, the desired dosing interval, and the desired dose.

System based output based on input and received by the system Various information, such as PK curves, PK parameters, such as incremental recovery (Cmax/dose), mean residence time, terminal t1/2, clearance, Vss, AUC/dose, dose and associated trough, and spacing and associated troughs.

For example, for evaluating individualized patient PK, the system can It is recommended that the user enter 2-3 optimized PK sampling time points. In this case, the system output can include a PK curve and one or more selected PK parameters, such as incremental recovery (Cmax/dose), mean residence time, terminal t1/2, clearance, Vss, AUC, and up to 1%. Or X% of the time, etc. For example, Figure 15.

As another example, to use the loss discussed in the previous paragraph Individual PK parameters are selected for individualized dosing regimens, (i) the dose selected for acute therapy can be increased based on the degree of plasma FIX activity entered by the user after the dose, and (ii) the dose selected for control can be selected. The desired dose to be administered based on the user may be based on the desired dosing interval entered by the user, or (iii) the interval selected for control. In the first case, the system can output a dose (IU) based on the patient's incremental recovery. For example, Figure 16. In the second case, the system output can be a table of doses and associated troughs (eg, x IU/kg, 1% trough, y IU/kg, 2% trough, etc.). For example, the upper part of Figure 17. In the third In this case, the system output can be a table of intervals and associated valleys (eg, x day, 1% valley bottom, y IU/kg, 2% valley bottom, etc.), such as the bottom of FIG.

Users may wish to enter no individualized PK data Use the system below. In this case, the drug delivery output will be individualized based on the median population rather than on a particular patient. For example, Figure 18. In this manner, the user enters, for example, body weight and age, and (i) the degree of plasma FIX activity is increased after the dose, (ii) the desired dosage interval for control, or (iii) the desired dose for control. In the first case, the system can output a dose. In the second case, the system can output dose and associated troughs. See Table 16. In the third case, the system can output the interval and the associated valley. See Table 16.

You can enter age to determine if the system is suitable for the patient because The current popPK model version is for patients 12 years of age and older.

In some embodiments, the system complies with patient privacy laws. In some embodiments, the system is encrypted, for example, with SSL. In some embodiments, the entered patient information is generated anonymously.

In some embodiments, the system includes a user assistance function.

The user can be, for example, a physician, a nurse or another health care practitioner.

In some embodiments, the method further comprises selecting a dosing regimen based on the output information of the system and administering rFIXFc to the patient according to the selected protocol.

Example 9. Association of bleeding trends with time at the target FIX activity level in patients with severe hemophilia B treated with rFIXFc

The goal of using clotting factor instead of prophylactic treatment in hemophilia patients has been to convert severe hemophilia, defined as an endogenous factor activity <1%, to moderate (1% to 5%) and mild ( 5% to 40%) of the disease. The increase in time spent at 1% FVIII activity may be associated with a total bleeding event and an increase in joint hemorrhage in patients with severe hemophilia A. This association of FIX activity in patients with severe hemophilia B has not been recorded to date. In this paper, we report the analysis of bleeding trends relative to FIX activity in the recently completed Phase 3 B-LONG study. The B-LONG study evaluated the pharmacokinetics (PK), safety, and efficacy of recombinant FIX Fc fusion protein (rFIXFc) in patients with severe hemophilia B. In short, the B-LONG study has four treatment teams: weekly prevention, customized prevention, paroxysmal treatment, and perioperative management. The corresponding median annual bleeding rates for the first three treatment teams were 2.95, 1.38, and 17.69, respectively.

Methods: Based on 12 subjects from the 1/2a study and 123 subjects from B-LONG (>12 years old) The activity-time profile collected for 52 weeks of treatment yielded a 3-compartment population PK model of rFIXFc. Individual post hoc PK parameters are then derived to construct a continuous FIX activity-time profile for each dose administered by the subject in the B-LONG over the course of the study. The cumulative time of each subject at the target 1%, 3%, and 5% FIX levels was calculated and corrected to obtain the annual time at the respective target FIX levels. Negative binomial regression model was used to evaluate the number of bleeding events (overall, spontaneous, traumatic, and joint) and annual time (days) at 1%, 3%, and 5% FIX activity in all subjects in B-LONG ) the association between. The model was adjusted for age, body mass index, baseline HIV and HCV status, FIX genotype, number of bleeding events in the 12 months prior to entry into the study, and time of study by each subject.

Results: Multivariate negative binomial return at 1% FIX activity Analysis of the analysis estimated that the overall bleeding event increased with increasing time (p < 0.001). However, the association is essentially driven by subjects with paroxysmal treatment. Contrary to the value of 0 days for subjects who were prevented or customized for prevention, the median time for subjects with paroxysmal treatment was 171 days at 1%, because of the customized PK-driven drug regimen. Designed to maintain a target valley above 1%. The association was consistent with the distribution of bleeding events, and in the subjects of paroxysmal treatment, most of the bleeding events occurred at a predicted FIX activity level of 1%. Since the distribution of FIX trough content in the subject to be controlled is predicted to be substantially in the range of 1% to 5%, the accumulation time is continued at 3% and 5% to repeat the analysis. Both analyses found a statistically significant increase in predicted bleeding events over time spent below the level of increase in individual FIX activity (Figures 20 and 21). A significant association of spontaneous bleeding, traumatic hemorrhage, or joint bleeding analyzed separately at all three target FIX activity levels was also observed. When compared between thresholds (1% vs. 3% vs. 5%), the predicted bleeding rate decreased significantly with increasing trough and the predicted no bleeding potential improved with increasing trough. In addition, at the respective target trough (1%, 3%, or 5%), the probability of joint bleeding increases significantly as time spent increases.

Conclusion: This is a demonstration of adolescents treated with rFIXFc and In adult subjects, the first study that was associated with an increase in the duration of the target therapeutic FIX activity (1%, 3%, or 5%) was associated with an increased tendency to hemorrhage and a reduced likelihood of bleeding. Based on population PK simulations of FIX activity, these findings confirm the importance of a minimum treatment threshold of 1% and provide additional support for establishing a prophylactic dosing regimen for patients with severe hemophilia B.

Example

E1. A pharmaceutical composition comprising: (a) a long-acting Factor IX polypeptide; (b) a carbohydrate mixture comprising sucrose and mannitol; (c) sodium chloride (NaCl); (d) L-histamine Acid; and (e) polysorbate 20 or polysorbate 80.

E2. The pharmaceutical composition of embodiment E1 comprising from about 1% (w/v) to about 2% (w/v) sucrose.

E3. The pharmaceutical composition of embodiment E2 comprising about 1.2% (w/v) sucrose.

E4. The pharmaceutical composition of embodiment E2 comprising about 1.7% (w/v) sucrose.

E5. The pharmaceutical composition of embodiment E1 comprising from about 10 mg/ml to about 20 mg/ml sucrose.

E6. The pharmaceutical composition of embodiment E5 comprising about 11.9 mg/ml sucrose.

E7. The pharmaceutical composition of embodiment E5, comprising about 16.7 Mg/ml sucrose.

The pharmaceutical composition according to any one of embodiments E1 to E7, which comprises from about 2% (w/v) to about 4% (w/v) mannitol.

E9. The pharmaceutical composition of embodiment E8 comprising about 2.4% (w/v) mannitol.

E10. The pharmaceutical composition of embodiment E8 comprising about 3.3% (w/v) mannitol.

E11. The pharmaceutical composition of any one of embodiments E1 to E7, comprising from about 20 mg/ml to about 40 mg/ml mannitol.

E12. The pharmaceutical composition of embodiment E11 comprising about 23.8 mg/ml mannitol.

E13. The pharmaceutical composition of embodiment E11 comprising about 33.3 mg/ml mannitol.

E14. The pharmaceutical composition of embodiment E1 comprising from about 1.0% to about 2.0% sucrose and from about 2.0% (w/v) to about 4.0% (w/v) mannitol.

E15. The pharmaceutical composition of embodiment E14 comprising about 1.2% (w/v) sucrose and about 2.4% (w/v) mannitol.

E16. The pharmaceutical composition of embodiment E14 comprising about 1.7% (w/v) sucrose and about 3.3% (w/v) mannitol.

E17. The pharmaceutical composition of embodiment E1 comprising from about 10 mg/ml to about 20 mg/ml sucrose and from about 20 mg/ml to about 40 mg/ml mannitol.

E18. The pharmaceutical composition of embodiment E17, comprising about 11.9 mg/ml sucrose and about 23.8 mg/ml mannitol.

E19. The pharmaceutical composition of embodiment E17, comprising about 16.7 mg/ml sucrose and about 33.3 mg/ml mannitol.

E20. A pharmaceutical combination according to any one of embodiments E1 to E19 A solution comprising between about 50 mM and about 60 mM NaCl.

E21. The pharmaceutical composition of embodiment E20, comprising about 55.6 mM NaCl.

E22. A pharmaceutical combination according to any one of embodiments E1 to E19 A solution comprising between about 3 mg/ml and about 4 mg/ml of NaCl.

E23. The pharmaceutical composition of embodiment E22, comprising about 3.25 mg/ml NaCl.

E24. A pharmaceutical combination according to any one of embodiments E1 to E23 A substance comprising between about 20 mM and about 40 mM L-histamine.

E25. The pharmaceutical composition of embodiment E24, comprising about 25 mM L-histamine.

E26. The pharmaceutical composition of embodiment E24, comprising about 35 mM L-histamine.

E27. A pharmaceutical combination according to any one of embodiments E1 to E23 And comprising L-histamine between about 3 mg/ml and about 6 mg/ml.

E28. The pharmaceutical composition of embodiment E27, comprising about 3.88 mg/ml L-histamine.

E29. The pharmaceutical composition of embodiment E27, comprising about 5.43 mg/ml L-histidine.

E30. A pharmaceutical combination according to any one of embodiments E1 to E29 And comprising polysorbate 20 or polysorbate 80 between about 0.008% (w/v) and about 0.020% (w/v).

E31. The pharmaceutical composition of embodiment E30 comprising about 0.010% (w/v) polysorbate 20 or polysorbate 80.

E32. The pharmaceutical composition of embodiment E30 comprising about 0.014% (w/v) polysorbate 20 or polysorbate 80.

The pharmaceutical composition according to any one of embodiments E1 to E29, which comprises between about 0.08 mg/ml and about 0.2 mg/ml of polysorbate 20 or polysorbate 80.

E34. The pharmaceutical composition of embodiment E33 comprising about 0.10% mg/ml polysorbate 20 or polysorbate 80.

E35. The pharmaceutical composition of embodiment E33 comprising about 0.14 mg/ml polysorbate 20 or polysorbate 80.

The pharmaceutical composition of any one of embodiments E1 to E35, wherein the long acting FIX polypeptide comprises human FIX fused to an FcRn binding partner.

The pharmaceutical composition of embodiment E36, wherein the long acting FIX polypeptide comprises an amino acid sequence comprising at least 90%, at least 95% or 100% identical to the amino acids 1 to 642 of SEQ ID NO: 2. a primary unit, and a second unit comprising an amino acid sequence that is at least 90% to 95% identical to the amino acids 1 to 227 of SEQ ID NO: 4.

E38. The pharmaceutical composition of any one of embodiments E1 to E37, comprising the long acting FIX polypeptide at a concentration between about 25 IU/ml and about 1200 IU/ml.

E39. The pharmaceutical composition of embodiment E38 comprising 50 IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml or 600 IU/ml of the long acting FIX polypeptide.

E40. The pharmaceutical composition of embodiment E39 comprising 50 IU/ml, 100 IU/ml, 200 IU/ml or 400 IU/ml of the long acting FIX polypeptide.

E41. The pharmaceutical composition of embodiment E39 comprising 600 IU/ml of the long acting FIX polypeptide.

E42. The pharmaceutical composition of embodiment E1 comprising: (a) between about 25 IU/ml and about 700 IU/ml of the long acting FIX polypeptide; (b) about 1% (w/v) and about 2% Sucrose between (w/v); (c) mannitol between about 2% (w/v) and about 4% (w/v); (d) NaCl between about 50 mM and about 60 mM; (e) between about 20 mM and about 40 mM of L-histamine; and (f) between about 0.008% (w/v) and about 0.015% of polysorbate 20 or polysorbate 80.

E43. The pharmaceutical composition of embodiment E42 comprising: (a) about 50 IU/ml of the long acting FIX polypeptide; (b) about 1.2% (w/v) sucrose; (c) about 2.4% (w/v Mannitol; (d) about 55.6 mM NaCl; (e) about 25 mM L-histamine; and (f) about 0.010% (w/v) polysorbate 20 or polysorbate 80.

E44. The pharmaceutical composition of embodiment E42 comprising: (a) about 100 IU/ml of the long acting FIX polypeptide; (b) about 1.2% (w/v) sucrose; (c) about 2.4% (w/v Mannitol; (d) about 55.6 mM NaCl; (e) about 25 mM L-histamine; and (f) about 0.010% (w/v) polysorbate 20 or polysorbate 80.

E45. The pharmaceutical composition of embodiment E42 comprising: (a) about 200 IU/ml of the long acting FIX polypeptide; (b) about 1.2% (w/v) sucrose; (c) about 2.4% (w/v Mannitol; (d) about 55.6 mM NaCl; (e) about 25 mM L-histamine; and (f) about 0.010% (w/v) polysorbate 20 or polysorbate 80.

E46. The pharmaceutical composition of embodiment E42 comprising: (a) about 400 IU/ml of the long acting FIX polypeptide; (b) about 1.2% (w/v) sucrose; (c) about 2.4% (w/v Mannitol; (d) about 55.6 mM NaCl; (e) about 25 mM L-histamine; and (f) about 0.010% (w/v) polysorbate 20 or polysorbate 80.

E47. The pharmaceutical composition of embodiment E42 comprising: (a) about 600 IU/ml of the long acting FIX polypeptide; (b) about 1.7% (w/v) sucrose; (c) about 3.3% (w/v) mannitol; (d) about 55.6 mM NaCl; (e) about 35 mM L-histamine; and (f) about 0.014% (w/v) polysorbate 20 or polysorbate 80.

E48. The pharmaceutical composition of embodiment E1 comprising: (a) between about 25 IU/ml and about 700 IU/ml of the long acting FIX polypeptide; (b) between about 10 mg/ml and about 20 mg/ml Sucrose; (c) mannitol between about 20 mg/ml and about 40 mg/ml; (d) between about 3 mg/ml and about 4 mg/ml NaCl; (e) about 3 mg/ml and about 6 mg/ml Between the L-histidine acid; and (f) between about 0.08 mg/ml and about 0.15 mg/ml of polysorbate 20 or polysorbate 80.

E49. The pharmaceutical composition of embodiment E48 comprising: (a) about 50 IU/ml of the long acting FIX polypeptide; (b) about 11.9 mg/ml sucrose; (c) about 23.8 mg/ml mannitol; d) about 3.25 mg/ml NaCl; (e) about 3.88 mg/ml L-histamine; and (f) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E50. The pharmaceutical composition of embodiment E48, comprising: (a) about 100 IU/ml of the long acting FIX polypeptide; (b) about 11.9 mg/ml sucrose; (c) about 23.8 mg/ml mannitol; (d) about 3.25 mg/ml NaCl; (e) about 3.88 mg/ml L-histamine; and (f) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E51. The pharmaceutical composition of embodiment E48, comprising: (a) about 200 IU/ml of the long acting FIX polypeptide; (b) about 11.9 mg/ml sucrose; (c) about 23.8 mg/ml mannitol; d) about 3.25 mg/ml NaCl; (e) about 3.88 mg/ml L-histamine; and (f) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E52. The pharmaceutical composition of embodiment E48, comprising: (a) about 400 IU/ml of the long acting FIX polypeptide; (b) about 11.9 mg/ml sucrose; (c) about 23.8 mg/ml mannitol; d) about 3.25 mg/ml NaCl; (e) about 3.88 mg/ml L-histamine; and (f) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E53. The pharmaceutical composition of embodiment E48, comprising: (a) about 600 IU/ml of the long acting FIX polypeptide; (b) about 16.7 mg/ml sucrose; (c) about 33.3 mg/ml mannitol; d) about 3.25 mg/ml NaCl; (e) about 5.43 mg/ml L-histamine; and (f) about 0.14 mg/ml polysorbate 20 or polysorbate 80.

E54. A medical kit comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) a long-acting FIX polypeptide, (ii) sucrose; (iii) mannitol; (iv) L - histidine; and (v) polysorbate 20 or polysorbate 80; and (b) a second comprising 0.325% (w/v) NaCl to be combined with the lyophilized powder of the first container container.

E55. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 250 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 50 IU/ml of the long acting FIX polypeptide; (ii) about 1.2% (w) /v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80.

E56. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 500 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 100 IU/ml of the long acting FIX polypeptide; (ii) about 1.2% (w) /v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; and (vi) about 0.01% (w/v Polysorbate 20 or polysorbate 80.

E57. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 1000 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; (b) a second container comprising, in combination with the lyophilized powder of the first container, in a volume sufficient to produce 0.325% (w/v) NaCl comprising: (i) about 200 IU/ml of the long-acting FIX a polypeptide; (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80.

E58. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 2000 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 400 IU/ml of the long acting FIX polypeptide; (ii) about 1.2% (w) /v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine; (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80.

E59. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 3000 IU of the long acting FIX polypeptide, (ii) about 83.3 mg sucrose; Iii) about 167 mg of mannitol; (iv) about 27.2 mg of L-histamine; and (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 600 IU/ml of the long acting FIX polypeptide; (ii) about 1.7% (w) /v) sucrose; (iii) about 3.3% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 35 mM L-histamine; and (vi) about 0.014% (w/v Polysorbate 20 or polysorbate 80.

E60. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 250 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; (b) comprising, in combination with the lyophilized powder of the first container, a second container having a volume sufficient to produce 0.325% (w/v) NaCl comprising: (i) about 50 IU/ml of the long-acting FIX a polypeptide; (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; Vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E61. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 500 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 100 IU/ml of the long acting FIX polypeptide; (ii) about 11.9 mg/ml Sucrose; (iii) about 23.8 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E62. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 1000 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder combination is in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 200 IU/ml of the long acting FIX polypeptide; (ii) about 11.9 mg/ml Sucrose; (iii) about 23.8 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; and (vi) about 0.10 mg/ml polysorbate Ester 20 or polysorbate 80.

E63. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 2000 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; Iii) about 119 mg of mannitol; (iv) about 19.4 mg of L-histamine; and (v) about 0.50 mg of polysorbate 20 or polysorbate 80; (b) a second container comprising, in combination with the lyophilized powder of the first container, in a volume sufficient to produce 0.325% (w/v) NaCl comprising: (i) about 400 IU/ml of the long-acting FIX a polypeptide; (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 3.88 mg/ml L-histamine; Vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80.

E64. The medical kit of embodiment E54, comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 3000 IU of the long acting FIX polypeptide, (ii) about 83.3 mg sucrose; Iii) about 167 mg of mannitol; (iv) about 27.2 mg of L-histamine; and (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder combination is in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 600 IU/ml of the long acting FIX polypeptide; (ii) about 16.7 mg/ml Sucrose; (iii) about 33.3 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 5.43 mg/ml L-histamine; (vi) about 0.14 mg/ml polysorbate 20 or polysorbate 80.

The kit of any one of embodiments E54 to E64, wherein the first container is a glass vial comprising a rubber stopper.

The kit of any one of embodiments E54 to E65, wherein the second container is a syringe body, and wherein the syringe body is accompanied by a plunger.

E67. The kit of embodiment E66, further comprising a joint that connects the glass vial to the syringe body.

E68. The kit of embodiment E66 or embodiment E67, further comprising an infusion tube with a needle adapted to be connected to the syringe for intravenous infusion.

E69. A method of administering a long-acting FIX polypeptide to a subject with hemophilia B in need of prevention, comprising administering intravenously to the subject at an initial dose of about 50 IU/kg once a week. The pharmaceutical composition of any one of embodiments E1 to E53, wherein the administration comprises preventing a bleeding event in the subject or reducing a frequency of bleeding events in the subject.

E70. A method of administering a long-acting FIX polypeptide to a subject with hemophilia B in need of prevention, comprising intravenously administering to the subject at an initial dose of about 100 IU/kg once every 10 to 14 days A pharmaceutical composition according to any one of embodiments E1 to E53, wherein the administering comprises preventing a bleeding event in the subject or reducing a frequency of bleeding events in the subject.

E71. The method of embodiment E69 or embodiment E70, wherein the control dose or dose frequency is subsequently adjusted based on the response of the subject.

E72. A need for treatment of mild to moderate bleeding events A method of administering a long-acting FIX polypeptide to a subject of hemophilia, comprising intravenously administering to the subject at any of the initial doses from about 30 IU/kg to about 60 IU/kg as in any of embodiments 1 to 53 A pharmaceutical composition wherein the administration controls, alleviates or reverses the bleeding event.

E73. The method of embodiment E72, further comprising Subjects exhibiting additional signs of bleeding, then one or more additional doses are administered every 48 hours.

E74. A type B hemophilia that requires treatment for severe bleeding episodes A method of administering a long-acting FIX polypeptide to a subject comprising administering to the subject intravenously a pharmaceutical composition according to any one of embodiments E1 to E53 at an initial dose of about 100 IU/kg, wherein the administration Control, moderate or reverse the bleeding event.

E75. The method of embodiment E74, further comprising Upon completion of the bleeding event, another dose of the pharmaceutical composition is administered at about 80 IU/kg after about 6 to 10 hours.

E76. The method of embodiment E75, further comprising Upon completion of the bleeding event, one or more additional doses of the pharmaceutical composition are administered at 80 IU/kg every 24 hours for three consecutive days.

E77. The method of embodiment E76, further comprising each One or more additional doses of the pharmaceutical composition are administered at 80 IU/kg for 48 hours until the bleeding event is controlled.

E78. A type of hemophilia B subject who needs surgery A method of administering a long-acting FIX polypeptide comprising administering to a subject of a hemophilia B who is undergoing minor surgery at a dose of between about 50 IU/kg and 80 IU/kg A pharmaceutical composition according to any one of embodiments E1 to E53, wherein the administration controls bleeding of the subject during and after surgery.

E79. The method of embodiment E78, further comprising To control post-operative bleeding, another dose of the pharmaceutical composition is administered at about 50 IU/kg to 80 IU/kg about 24 to about 48 hours after surgery.

E80. A type of hemophilia B subject who needs surgery A method of administering a long-acting FIX polypeptide comprising intravenously administering a pharmaceutical combination of any one of embodiments E1 to E53 to a subject with hemophilia B who is undergoing major surgery at a dose of about 100 IU/kg The subject, wherein the administration controls bleeding of the subject during and after surgery.

E81. The method of embodiment E80, further comprising To control post-operative bleeding, another dose of the pharmaceutical composition is administered at about 80 IU/kg after about 6 to 10 hours.

E82. The method of embodiment E81, further comprising To control post-operative bleeding, one or more additional doses of the pharmaceutical composition were administered at 80 IU/kg every 24 hours for three days.

E83. The method of embodiment E82, further comprising To control post-operative bleeding, one or more additional doses of the pharmaceutical composition are administered at 80 IU/kg every 48 hours.

E84. The method of any one of embodiments E69 to E82, The long-acting FIX polypeptide of the desired dose can be obtained from a single medical kit of any of embodiments 54 to 68.

E85. The method of any one of embodiments E69 to E82, The long-acting FIX polypeptide of the desired dose can be used in Examples 54 to 68. Two or more medical kits of either are obtained, and wherein the contents of the two or more medical kits are combined prior to administration.

The pharmaceutical composition of any one of embodiments E1 to E53, wherein after a single intravenous infusion of 50 IU/kg of the long acting FIX polypeptide, the average T _{1/2 β} (activity) of the long acting FIX polypeptide is about 70 hours to about 95 hours.

The pharmaceutical composition of any one of embodiments E1 to E53, wherein the average T _{1/2 β} (activity) is about 82 hours.

E88. As in Example E86 or a pharmaceutical composition Example E87 of the embodiment, wherein after the infusion of 50IU / kg BENEFIX ^® in a single intravenous, the average _{T 1 /} 2β (active) than the polypeptide consisting of full length mature Factor IX composition of (BENEFIX ^® ) at least about 2 times to about 3 times higher.

E89. The pharmaceutical composition of embodiment E88, wherein the average T _{1/2 β} (activity) is about 2.4 times higher than the polypeptide consisting of full length mature factor IX (BENEFIX ^® ).

The pharmaceutical composition of any one of embodiments E1 to E53 or E86 to E89, wherein the average _Cmax of the long acting FIX polypeptide is about 30 IU after a single intravenous infusion of 50 IU/kg of the long acting FIX polypeptide. /dL to about 50 IU/dL.

E91. The pharmaceutical composition of embodiment E90, wherein the average _Cmax is about 40.8 IU/dL.

E92. Any of Embodiments E1 to E53 or E86 to E91 Pharmaceutical composition wherein the average area under the curve of the long acting FIX polypeptide after a single intravenous infusion of 50 IU/kg of the long acting FIX polypeptide (AUC/dose) is from about 27 IU*h/dL/IU/kg to about 35 IU*h/dL/IU/kg.

E93. The pharmaceutical composition of embodiment E92, wherein the average AUC/agent is about 31.32 IU*h/dL/IU/kg.

After E94. As in Example E92 or embodiment E93 of the pharmaceutical composition embodiments, wherein the infusion polypeptide from the full length mature Factor IX composition of (BENEFIX ^®) in 50IU / kg single intravenous, the average AUC / agent than the full length mature Factor The polypeptide consisting of IX (BENEFIX ^® ) is at least about 1.8 times to about 2.1 times higher.

E95. E94 as described in Example embodiments of the pharmaceutical composition, wherein the mean AUC / dose, about 1.99 times higher than the polypeptide (BENEFIX ^®) composed of the full length mature Factor IX.

E96. A method of reducing annual bleeding rate in a subject with hemophilia B, comprising administering a fixed or individualized dose at regular or individualized dosing intervals as in Examples 1 to 53 and 86 to 95 A pharmaceutical composition of any of the above.

The method of embodiment E96, wherein the pharmaceutical composition is administered to the subject at a fixed dosing interval by a fixed dose.

The method of embodiment E97, wherein the fixed dose of the pharmaceutical composition is about 50 IU/kg, and the fixed dosing interval is about one week.

The method of embodiment E97, wherein the fixed dose of the pharmaceutical composition is about 100 IU/kg, and the fixed dosing interval is between about 10 days and about 14 days.

E100. The method of embodiment E96, wherein the pharmaceutical combination The lines are administered to the subject at a fixed dosing interval at individualized doses.

E101. The method of embodiment E100, wherein the fixed administration The interval is about one week.

E102. The method of embodiment E100 or embodiment E101, The individualized dose is adjusted to achieve a plasma FIX trough activity level between about 1 IU/dl and about 3 IU/dl.

E103. The method of any one of embodiments 101 to 102, The individualized dose administered about once a week is between about 30 IU/kg and about 60 IU/kg.

E104. The method of embodiment E103, wherein about weekly is administered The individualized dose is once between about 32 IU/kg and about 54 IU/kg.

E105. The method of embodiment E104, wherein about weekly is administered The average individualized dose at one time was about 41 IU/kg.

E106. The method of any one of embodiments E100 to E105 The method wherein the subject's annual bleeding rate is reduced by about 70% to about 90% compared to the average annual bleeding rate of a type B hemophilia subject treated by paroxysmal or on-demand administration.

E107. The method of embodiment E106, wherein the subject The annual bleeding rate is reduced by about 76% to about 89%.

E108. The method of embodiment E107, wherein the subject The annual bleeding rate is reduced by approximately 83%.

The method of embodiment E96, wherein the pharmaceutical combination The system is administered to the subject at a fixed dose at an individualized dosing interval.

E110. The method of embodiment E109, wherein the fixed dose It is about 100 IU/kg.

E111. The method of embodiment E109 or embodiment E110, The individualized dosing interval is adjusted to achieve a plasma FIX trough activity level between about 1 IU/dl and about 3 IU/dl.

E112. The method of any one of embodiments E109 to E111 The method wherein the individualized dosing interval is between about 10 days and about 14 days.

E113. The method of embodiment E112, wherein the individualizing The drug interval is about 13 days.

E114. The method of any one of embodiments E109 to E113 The method wherein the subject's annual bleeding rate is reduced by about 80% to about 95% compared to the average annual bleeding rate of a type B hemophilia subject treated by paroxysmal or on-demand administration.

E115. The method of embodiment E114, wherein the subject The annual bleeding rate is reduced by about 80% to about 92%.

E116. The method of embodiment E115, wherein the subject The annual bleeding rate is reduced by approximately 87%.

E117. As in Examples E1 to E53 and E86 to E95 A pharmaceutical composition further comprising a short acting FIX polypeptide.

E118. The pharmaceutical composition of embodiment E117, wherein the short The FIX polypeptide comprises wild type FIX or consists of wild type FIX.

E119. as in embodiments E1 to E53, E86 to E95, and A pharmaceutical composition according to any one of E117 and E118 which is a liquid formulation.

E120. as in embodiments E1 to E53, E86 to E95, and A pharmaceutical composition according to any one of E117 and E118 which is a lyophilized powder.

E121. as in embodiments E1 to E53, E86 to E95, and A pharmaceutical composition according to any one of E117 and E118 which is a suspension.

E122. A vial comprising as in embodiment E119 A pharmaceutical composition according to any one of E121.

E123. A cartridge comprising the medicament of embodiment E119 combination.

E124. A syringe comprising as in embodiment E119 or A pharmaceutical composition of E120.

E125. The syringe of embodiment E124, which is a dual chamber injection Device.

E126. The kit of any of embodiments E54 to E68, Wherein the second container contains a preservative in an amount sufficient to provide antimicrobial activity.

E127. The pharmaceutical composition of embodiment E119, wherein the liquid The body conditioning formulation further comprises a preservative in an amount sufficient to provide antimicrobial activity.

E128. The kit of embodiment E126 or as claimed The pharmaceutical composition according to Item 127, wherein the preservative is selected from the group consisting of phenol, m-cresol, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, phenoxyethanol , any other pharmaceutically acceptable preservative, and any combination thereof.

The pharmaceutical composition of any one of embodiments E126 or E128, or the pharmaceutical composition of embodiment E127 or E128, wherein the preservative is benzyl alcohol.

E130. The kit or pharmaceutical composition of embodiment E129, wherein the concentration of benzyl alcohol is between 0.5% and 0.9%.

E131. A method of estimating information on administration of rFIXFc that is individualized to a patient, the method comprising: (a) receiving a patient by a computer-based system comprising the rFIXFc population pharmacokinetic (popPK) model of Example 6 and a Bayesian estimation program (b) using the popPK model, the Bayesian estimation program and the received information, calculating the individualized rFIXFc administration information by the computer-based system, and (c) by using the popPK model, the Bayesian estimation program, and the received information The computer based system outputs the individualized dosing information.

E132. The method of embodiment E131, further comprising selecting a dosing regimen based on the individualized dosing information of the output of (c) and administering to the patient rFIXFc according to the selected dosing regimen.

E133. A computer readable storage medium having stored thereon instructions that, when executed by a processor, cause the processor to perform the method of embodiment E131.

E134. A system comprising a processor and a memory having instructions stored by the processor to cause the processor to perform the method of embodiment E131.

E135. A method for estimating a rFIXFc dosing regimen based on a median popPK, the method comprising: (a) receiving patient information and a desired therapeutic result by a computer-based system comprising the rFIXFc popPK model of Example 6 and a Bayesian estimation program At least one of the information, (b) using the popPK model, the Bayesian estimation program and the received information, calculating the median rFIXFc PK information by the computer based system, and (c) outputting by the computer based system The median PK information.

E136. The method of embodiment E135, further comprising selecting a dosing regimen based on the median PK information of the output of (c), and administering rFIXFc to the patient according to the selected dosing regimen.

E137. A computer readable storage medium having stored thereon instructions that, when executed by a processor, cause the processor to perform the method of embodiment E135.

E138. A system comprising a processor and a memory having instructions stored by the processor to cause the processor to perform the method of embodiment E135.

E139. A method of estimating an individual patient rFIXFc PK, the method comprising: (a) receiving individual rFIXFc PK information by a computer-based system comprising the rFIXFc population pharmacokinetic (popPK) model of Example 6 and a Bayesian estimation program; Using the popPK model, the Bayesian estimation program and the received information, estimating the individualized patient rFIXFc PK information by the computer based system, and (c) outputting the personalized patient PK information by the computer based system.

E140. The method of embodiment E139, further comprising selecting an administration protocol for the individualized patient PK information based on the output of (c), and administering to the patient rFIXFc according to the selected protocol.

E141. A computer readable storage medium having stored thereon instructions that, when executed by a processor, cause the processor to perform the method of embodiment E139.

E142. A system comprising a processor and a memory having instructions stored by the processor to cause the processor to perform the method of embodiment E139.

E143. The method of embodiment 131, wherein the information to be treated is that the level of plasma FIX activity is increased after administration and the output information is a dose for acute treatment.

E144. The method of embodiment 131, wherein the information to be treated is the desired dosing interval and the output information is the dose for control.

E145. The method of embodiment 131, wherein the information to be treated is the desired dose and the output information is an interval for control.

E146. The method of embodiment 139, wherein (a) further comprises receiving other patient information by the computer based system.

The method of any one of embodiments 131, 135, or 146, wherein the patient information is age or weight.

The above description of the specific embodiments will fully disclose the general nature of the invention so that others can be easily utilized without the undue experimentation Modifications and/or adaptations of these specific embodiments are made for various applications. Therefore, the adaptations and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments. It is understood that the phraseology or terminology herein is used for the purposes of the description and

<110> Baijian Aike Mazhou Co., Ltd. Pierce Green Trux Shanmansha Peters Robert Jiang Haiyan Bleidel Mark

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<400> 4

Claims (130)

A method of administering long acting factor IX (FIX) to a human subject in need thereof, comprising administering to the subject from about 10 IU/kg to about 200 IU at a dosing interval of about one week or more. A kg dose of the long acting FIX polypeptide. The method of claim 1, wherein the subject is in need of treatment for a bleeding disorder. The method of claim 1 or 2, wherein the dosage of the long acting FIX polypeptide is from about 10 IU/kg to about 50 IU/kg, from about 10 IU/kg to about 100 IU/kg, from about 25 IU/kg to about 50 IU/ Kg, from about 25 IU/kg to about 75 IU/kg, from about 25 IU/kg to about 100 IU/kg, from about 25 IU/kg to about 125 IU/kg, from about 25 IU/kg to about 150 IU/kg, from about 50 IU/kg to about 100 IU/ Kg, from about 50 IU/kg to about 150 IU/kg, from about 100 IU/kg to about 150 IU/kg, from about 150 IU/kg to about 200 IU/kg, or any combination thereof. The method of any one of claims 1 to 3 wherein the dosage of the long acting FIX polypeptide is for controlling one or more bleeding events. The method of claim 4, wherein the dosage is about 50 IU/kg. The method of claim 4, wherein the annual bleeding rate of the bleeding events is less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, or less than 10. The method of any one of claims 1 to 3, wherein The dose of the long acting FIX polypeptide is used to control one or more bleeding events in an individualized interval. The method of claim 7, wherein the dosage of the long acting FIX polypeptide is about 100 IU/kg. The method of claim 7 or 8, wherein the annual bleeding rate of the bleeding events is less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, or less than 9. The method of any one of claims 1 to 3 wherein the dosage of the long acting FIX polypeptide is for the treatment of one or more bleeding events as needed. The method of claim 10, wherein the annual bleeding rate of the bleeding event is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20 , less than 21, less than 22, less than 23, less than 24, less than 25 or less than 26. The method of any one of claims 1 to 3, wherein the dose of the long acting FIX polypeptide is for perioperative management of a bleeding disorder. The method of any one of claims 1 to 12, wherein the dose is administered at a frequency of administration from about once a week to about once a month. The method of claim 13, wherein the frequency of administration is about once a week, once in about two weeks, twice in about one month, once in about three weeks, once in about four weeks, or once in about one month. The method of claim 14, wherein the frequency of administration is about once a week. The method of claim 14, wherein the frequency of administration is once in about two weeks or twice in about one month. The method of any one of claims 1 to 14, wherein the dosing interval is about every 5 days, about every 6 days, about every 7 days, about every 8 days, about every 9 days, about every 10 hours. Every day, about every 11 days, about every 12 days, about every 13 days, about every 14 days, about every 15 days, about every 16 days, about every 17 days, about every 18 days, about every 19 days, about every 20 days Day or about every 21 days. The method of claim 17, wherein the dosing interval is every 7 to 14 days. The method of any one of claims 1 to 18, wherein the long-acting FIX polypeptide has a T _{1/2 β} (activity) of at least about 40 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours. At least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours At least about 180 hours or at least about 190 hours. The method of claim 19, wherein the T _1/2β (activity) is from about 40 hours to about 193 hours, from about 50 hours to about 203 hours, from about 30 hours to about 183 hours, from about 40 hours to about 203. Hour, about 40 hours to about 213 hours, about 40 hours to about 223 hours, about 30 hours to about 213 hours, about 30 hours to about 223 hours, about 50 hours to about 213 hours, about 50 hours to about 223 hours, From about 60 hours to about 203 hours, or from about 60 hours to about 213 hours. The method of claim 17 or 18, wherein the T _{1/2 β} (activity) is from about 40 hours to about 193 hours. The method of any one of claims 19 to 21, wherein the average of the T _{1/2 β} (activity) is about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, or about 92 hours. The scope of the patent application method of any one of item 19 to 22, wherein the _{T 1 /} 2β (activity) of the average ratio of SEQ ID NO: 2 amino acids 1-415 of high or polypeptide consisting of at least BENEFIX ^® About 2.0 times. The scope of the patent application method of any one of 23, wherein the _{T 1 /} 2β (activity) of the average ratio of SEQ ID NO: 2 amino acids 1-415 of the polypeptide or BENEFIX ^® composition is at least about 2.0 times At least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about 2.9 times, at least about 3.0 times At least about 3.1 times or at least about 3.2 times. The method of any one of claims 1 to 24, wherein the long-acting FIX polypeptide has a T _{1/2 β} (antigen) of at least about 60 hours, at least about 80 hours, at least about 100 hours, at least about 120 hours. At least about 140 hours, at least about 160 hours, at least about 180 hours, at least about 200 hours, at least about 220 hours, at least about 240 hours, at least about 260 hours, at least about 280 hours, at least about 300 hours, at least about 320 hours At least about 340 hours, at least about 360 hours, or at least about 370 hours. The method of claim 25, wherein the T _1/2β (antigen) is from about 63 hours to about 372 hours, from about 63 hours to about 382 hours, from about 63 hours to about 392 hours, from about 63 hours to about 402. Hours, from about 63 hours to about 412 hours, from about 53 hours to about 372 hours, from about 53 hours to about 382 hours, from about 53 hours to about 392 hours, from about 53 hours to about 402 hours, from about 53 hours to about 513 hours, From about 70 hours to about 372 hours, from about 70 hours to about 382 hours, from about 70 hours to about 392 hours, from about 70 hours to about 402 hours, or from about 70 hours to about 412 hours. The method of claim 25 or 26, wherein the T _1/2β (antigen) is from about 63 hours to about 372 hours. The method of any one of claims 25 to 27, wherein the average of the T _1/2β (antigen) is about 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, about 120 hours, about 121 hours, about 122 hours, about 123 hours, about 124 hours, about 125 hours, about 126 hours, about 127 hours, about 128 hours, about 129 hours, about 130 hours, or about 131 hours. The scope of the patent application of the method of any one of 25-28, wherein the _{T 1 /} 2β (antigen) by the average of the ratio of SEQ ID NO: 2 amino acids 1-415 of high or polypeptide consisting of at least BENEFIX ^® About 3.0 times. The scope of the patent application method of any one of 39, wherein the _{T 1 /} 2β (antigen) by the average of the ratio of SEQ ID NO: 2 amino acids 1-415 of the polypeptide or BENEFIX ^® composition is at least about 2.0 times At least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times. The method of any one of claims 1 to 30, further comprising measuring a baseline FIX activity of the subject prior to administering the long acting FIX polypeptide. The method of any one of claims 1 to 31, wherein after the administration, the plasma trough content of the long-acting FIX polypeptide in the subject is maintained at about 1% above the baseline. The method of claim 32, wherein the plasma trough content of the long-acting FIX polypeptide in the subject is maintained between about 1% and about 5%, between about 1% and about 6%, above about baseline, about Between 1% and about 7%, between about 1% and about 8%, between about 1% and about 9%, between about 1% and about 10%, between about 1% and about 11%, about Between 1% and about 12%, between about 1% and about 13%, between about 1% and about 14%, between about 1% and about 15%. The method of any one of claims 1 to 33, wherein the dose comprises less than 25% of the long-acting Factor IX polypeptide in a fully phosphorylated state and less than 25% of the long-acting state in a fully sulfated state. Factor IX polypeptide. The method of claim 34, wherein the dose comprises less than about 10% of the long-acting Factor IX polypeptide in a phosphorylated state and small About 9% of the long-acting Factor IX polypeptide in the sulfated state. The method of any one of claims 1 to 35, wherein the dose is about 50 IU/kg and the dosing interval is about 7 days. The method of any one of claims 1 to 35, wherein the dose is about 100 IU/kg and the dosing interval is at least about 14 days. The method of any one of claims 1 to 35, wherein the dose is about 150 IU/kg and the dosing interval is at least about 21 days. The method of any one of claims 1 to 38, wherein the long acting FIX polypeptide is a chimeric polypeptide comprising a FIX polypeptide and an FcRn binding partner. The method of claim 39, wherein the FcRn binding partner comprises an Fc region. The method of claim 39, wherein the long acting FIX polypeptide further comprises a second FcRn binding partner. The method of claim 41, wherein the second FcRn binding partner comprises a second Fc region. The method of claim 41, wherein the FcRn binding partner is associated with the second FcRn binding partner. The method of claim 43, wherein the association is a covalent bond. The method of claim 44, wherein the covalent bond is a disulfide bond. The method of any one of claims 41 to 45, wherein the second FcRn binding partner is not linked to the amino acid sequence by a peptide bond Column. The method of any one of claims 1 to 46, wherein the long acting FIX polypeptide is a FIX monomer dimer hybrid. The method of any one of claims 1 to 47, wherein the subject is in need of control or prevention of a bleeding or bleeding event. The method of claim 48, wherein the subject needs to control or prevent bleeding in the following: a small amount of blood loss, joint blood, superficial muscle loss, soft tissue blood loss, moderate blood loss, intramuscular or soft tissue accompanying dissection Blood loss, mucosal blood loss, hematuria, massive blood loss, pharyngeal blood loss, posterior pharyngeal blood loss, retroperitoneal blood loss, central nervous system blood loss, abrasion, incision, scratch, joint blood loss, nose bleeding, oral bleeding, bleeding gums, intracranial hemorrhage, Intraperitoneal hemorrhage, a small amount of spontaneous blood loss, post-traumatic bleeding, moderate skin abrasions, or spontaneous blood loss to the joints, muscles, internal organs, or brain. The method of claim 48, wherein the subject requires perioperative management. The method of claim 48 or 49, wherein the subject is required to manage bleeding associated with surgery or tooth extraction. The method of claim 48, wherein the subject will experience, is undergoing or has undergone major surgery. For example, the method of claim 52, wherein the major surgery is orthopedic surgery, large-area oral surgery, urinary surgery or hernia surgery. The method of claim 52, wherein the orthopedic surgery is to replace a knee, a hip or other major joint. The method of any one of claims 1 to 54 wherein the subject requires prophylactic treatment. The method of any one of claims 1 to 54 wherein the subject is in need of treatment as needed. The method of any one of claims 1 to 54 wherein the subject is in need of treatment for a bleeding event. For example, the method of claim 57, wherein the subject needs to treat joint blood, muscle bleeding, oral bleeding, blood loss, blood loss to the muscle, oral blood loss, trauma, head trauma, gastrointestinal bleeding, intracranial blood loss, Intra-abdominal blood loss, intrathoracic blood loss, fracture, central nervous system hemorrhage, hemorrhage in the posterior pharyngeal space, hemorrhage in the retroperitoneal space, or hemorrhage in the iliopsoas. For example, the method of claim 58 wherein the subject requires surgery, perioperative management, or surgery. For example, the method of claim 59, wherein the operation is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia incision, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery , intracranial surgery, intra-abdominal surgery, intrathoracic surgery or joint replacement surgery. The method of any one of claims 39 to 60, wherein the FIX polypeptide in the chimeric polypeptide is human Factor IX. The method of any one of claims 39 to 61, wherein the FIX polypeptide in the chimeric polypeptide is Mutant Factor IX. The method of any one of claims 39 to 62, wherein the FcRn binding partner in the chimeric polypeptide is a human Fc. The method of any one of claims 39 to 63, Wherein the FcRn binding partner in the chimeric polypeptide is a mutant Fc. The method of claim 64, wherein the FIX polypeptide is at least 90% different from the signalless sequence shown in Table 8A and the FIX amino acid sequence of the propeptide (amino acid 1 to 415 of SEQ ID NO: 2). Or 95% consistent. The method of claim 63, wherein the Factor IX is identical to the signal-free sequence shown in Table 8A and the Factor IX amino acid sequence of the propeptide (amino acid 1 to 415 of SEQ ID NO: 2). The method of any one of claims 40 to 66, wherein the Fc is at least Fc amino acid sequence (amino acid 1 to 227 of SEQ ID NO: 4) having no signal sequence as shown in Table 8B. 90% or 95% consistent. The method of claim 67, wherein the Fc is identical to the Fc amino acid sequence of the signalless sequence shown in Table 8B (amino acids 1 to 227 of SEQ ID NO: 4). The method of any one of claims 41 to 68, wherein the second FcRn binding partner in the chimeric polypeptide is a human Fc. The method of any one of claims 41 to 68, wherein the second FcRn binding partner in the chimeric polypeptide is a mutant Fc. The method of claim 69 or 70, wherein the Fc is at least 90% or 95 with the Fc amino acid sequence of the signalless sequence shown in Table 8B (amino acids 1 to 227 of SEQ ID NO: 4). % is consistent. The method of claim 71, wherein the Fc is identical to the Fc amino acid sequence of the signalless sequence shown in Table 6B (amino acids 1 to 227 of SEQ ID NO: 4). The method of any one of claims 40 to 72, Wherein the chimeric polypeptide comprises at least 90% or 95% identical to the signalless sequence shown in Table 8A and the Factor IX and Fc amino acid sequence of the propeptide (amino acids 1 to 642 of SEQ ID NO: 2). sequence. The method of claim 73, wherein the chimeric polypeptide comprises the Factor IX and Fc amino acid sequence (SEQ ID NO: 2 amino acid 1) having no signal sequence and propeptide as shown in Table 8A. To 642) a consistent sequence. The method of any one of claims 1 to 74, wherein the subject requires long-term treatment. The method of any one of claims 1 to 75, wherein the long-acting FIX polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. The method of any one of claims 1 to 76, wherein the dose is administered intravenously or subcutaneously. The method of any one of claims 19 to 77, wherein the T _1/2β (activity) is measured by single-stage coagulation analysis. The method of any one of claims 1 to 78, wherein the long-acting FIX polypeptide has an average residence time (MRT) of at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, At least about 90 hours, at least about 100 hours, at least about 110 hours, at least about 120 hours, at least about 130 hours, at least about 140 hours, at least about 150 hours, at least about 160 hours, at least about 170 hours, at least about 180 hours, or At least about 190 hours. For example, the method of claim 79, wherein the MRT is From about 50 hours to about 200 hours, from about 60 hours to about 210 hours, from about 60 hours to about 183 hours, from about 70 hours to about 150 hours, from about 70 hours to about 140 hours, from about 70 hours to about 130 hours, from about 80 hours Hours to about 120 hours, from about 80 hours to about 110 hours, or from about 88 hours to about 110 hours. The scope of the patent application method of any one of item 19 to 22, wherein the ratio of the MRT SEQ ID NO: 2 amino acids 1-415 of the polypeptide or BENEFIX ^® composition is at least about 2.0 times. The method of any one of claims 1 to 81, wherein the long-acting FIX polypeptide is formulated in the form of a pharmaceutical composition comprising: (a) the long-acting FIX polypeptide; (b) comprising sucrose and mannose a carbohydrate mixture of alcohol; (c) sodium chloride (NaCl); (d) L-histamine; and (e) polysorbate 20 or polysorbate 80. The method of claim 82, wherein the pharmaceutical composition comprises from about 1% (w/v) to about 2% (w/v) sucrose. The method of claim 83, wherein the pharmaceutical composition comprises about 1.2% (w/v) sucrose or about 1.7% (w/v) sucrose. The method of claim 82, wherein the pharmaceutical composition comprises from about 10 mg/ml to about 20 mg/ml sucrose. The method of claim 85, wherein the pharmaceutical composition comprises about 11.9 mg/ml sucrose or about 16.7 mg/ml sucrose. The method of any one of claims 82 to 86, wherein the pharmaceutical composition comprises from about 2% (w/v) to about 4% (w/v) mannitol. The method of claim 87, wherein the pharmaceutical composition comprises about 2.4% (w/v) mannitol or about 3.3% (w/v) mannitol. The method of any one of claims 82 to 86, wherein the pharmaceutical composition comprises from about 20 mg/ml to about 40 mg/ml mannitol. The method of claim 89, wherein the pharmaceutical composition comprises about 23.8 mg/ml mannitol or about 33.3 mg/ml mannitol. The method of claim 82, wherein the pharmaceutical composition comprises from about 1.0% to about 2.0% sucrose and from about 2.0% (w/v) to about 4.0% (w/v) mannitol. The method of claim 91, wherein the pharmaceutical composition comprises about 1.2% (w/v) sucrose and about 2.4% (w/v) mannitol. The method of claim 92, wherein the pharmaceutical composition comprises about 1.7% (w/v) sucrose and about 3.3% (w/v) mannitol. The method of claim 82, wherein the pharmaceutical composition comprises from about 10 mg/ml to about 20 mg/ml sucrose and from about 20 mg/ml to about 40 mg/ml mannitol. The method of claim 94, wherein the pharmaceutical composition comprises (i) about 11.9 mg/ml sucrose and about 23.8 mg/ml mannitol. Or (ii) about 16.7 mg/ml sucrose and about 33.3 mg/ml mannitol. The method of any one of claims 82 to 95, wherein the pharmaceutical composition comprises between about 50 mM and about 60 mM NaCl. The method of claim 96, wherein the pharmaceutical composition comprises about 55.6 mM NaCl. The method of any one of claims 82 to 95, wherein the pharmaceutical composition comprises between about 3 mg/ml and about 4 mg/ml of NaCl. The method of claim 98, wherein the pharmaceutical composition comprises about 3.25 mg/ml NaCl. The method of any one of claims 82 to 99, wherein the pharmaceutical composition comprises between about 20 mM and about 40 mM of L-histamine. The method of claim 100, wherein the pharmaceutical composition comprises about 25 mM L-histamine or about 35 mM L-histamine. The method of any one of claims 82 to 99, wherein the pharmaceutical composition comprises between about 3 mg/ml and about 6 mg/ml of L-histamine. The pharmaceutical composition comprises about 3.88 mg/ml L-histamine or about 5.43 mg/ml L-histamine, as in the method of claim 102. The method of any one of claims 82 to 103, wherein the pharmaceutical composition comprises about 0.008% (w/v) and about 0.020% Polysorbate 20 or polysorbate 80 between (w/v). The method of claim 104, wherein the pharmaceutical composition comprises about 0.010% (w/v) polysorbate 20 or polysorbate 80 or about 0.014% (w/v) polysorbate 20 or Polysorbate 80. The method of any one of claims 82 to 103, wherein the pharmaceutical composition comprises between about 0.08 mg/ml and about 0.2 mg/ml of polysorbate 20 or polysorbate 80. The method of claim 106, wherein the pharmaceutical composition comprises about 0.10% mg/ml polysorbate 20 or polysorbate 80 or about 0.14 mg/ml polysorbate 20 or polysorbate 80. . The method of any one of claims 1 to 35, wherein the rFIXFc polypeptide comprises a first amino acid sequence comprising at least 90% or 95% identical to the amino acids 1 to 642 of SEQ ID NO: 2. a subunit, and a second unit comprising an amino acid sequence that is at least 90% to 95% identical to the amino acids 1 to 227 of SEQ ID NO: 4. The method of claim 108, wherein the rFIXFc polypeptide comprises a first unit comprising amino acids 1 to 642 of SEQ ID NO: 2, and a first unit comprising amino acids 1 to 227 of SEQ ID NO: Secondary unit. The method of any one of claims 82 to 109, wherein the long acting FIX polypeptide is present at a concentration of between about 25 IU/ml and about 1200 IU/ml. The method of claim 110, wherein the pharmaceutical composition comprises 50 IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml. Or 600 IU/ml of the long acting FIX polypeptide. The method of claim 111, wherein the pharmaceutical composition comprises 50 IU/ml, 100 IU/ml, 200 IU/ml or 400 IU/ml of the long acting FIX polypeptide. The method of claim 111, wherein the pharmaceutical composition comprises 600 IU/ml of the long acting FIX polypeptide. The method of claim 82, wherein the pharmaceutical composition comprises: (a) between about 25 IU/ml and about 700 IU/ml of the long acting FIX polypeptide; (b) about 1% (w/v) and About 2% (w/v) of sucrose; (c) about 2% (w/v) and about 4% (w/v) of mannitol; (d) between about 50 mM and about 60 mM NaCl (e) between about 20 mM and about 40 mM of L-histamine; and (f) between about 0.008% (w/v) and about 0.015% of polysorbate 20 or polysorbate 80 . The method of claim 114, wherein the pharmaceutical composition comprises: (a) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml or 400 IU/ml of the long acting FIX polypeptide; (b) about 1.2% (w/v) sucrose; (c) about 2.4% (w/v) mannitol; (d) about 55.6 mM NaCl; (e) about 25 mM L-histamine; (f) about 0.010% (w/v) polysorbate 20 or polysorbate 80. The method of claim 114, wherein the pharmaceutical composition comprises: (a) about 600 IU/ml of the long acting FIX polypeptide; (b) about 1.7% (w/v) sucrose; (c) about 3.3% ( w/v) mannitol; (d) about 55.6 mM NaCl; (e) about 35 mM L-histamine; and (f) about 0.014% (w/v) polysorbate 20 or polysorbate 80 . The method of claim 82, wherein the pharmaceutical composition comprises: (a) between about 25 IU/ml and about 700 IU/ml of the long-acting FIX polypeptide; (b) about 10 mg/ml and about 20 mg/ml. Between the sucrose; (c) between about 20 mg/ml and about 40 mg/ml of mannitol; (d) between about 3 mg/ml and about 4 mg/ml of NaCl; (e) about 3 mg/ml and about L-histidine acid between 6 mg/ml; and (f) polysorbate 20 or polysorbate 80 between about 0.08 mg/ml and about 0.15 mg/ml. The method of claim 117, wherein the pharmaceutical composition comprises: (a) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml or about 400 IU/ml of the long-acting FIX polypeptide; (b) about 11.9 Mg/ml sucrose; (c) about 23.8 mg/ml mannitol; (d) about 3.25 mg/ml NaCl; (e) about 3.88 mg/ml L-histamine; and (f) about 0.10 mg/ml polysorbate 20 Or polysorbate 80. The method of claim 117, wherein the pharmaceutical composition comprises: (a) about 600 IU/ml of the long-acting FIX polypeptide; (b) about 16.7 mg/ml sucrose; (c) about 33.3 mg/ml mannose Alcohol; (d) about 3.25 mg/ml NaCl; (e) about 5.43 mg/ml L-histamine; and (f) about 0.14 mg/ml polysorbate 20 or polysorbate 80. The method of any one of claims 1 to 81, wherein the long-acting FIX polypeptide is packaged in a pharmaceutical kit comprising: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i a long-acting FIX polypeptide, (ii) sucrose; (iii) mannitol; (iv) L-histamine; and (v) polysorbate 20 or polysorbate 80; and (b) A second container of 0.325% (w/v) NaCl in combination with the lyophilized powder of the first container. For example, the method of claim 120, wherein the medical kit includes: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 250 IU, about 500 IU, about 1000 IU or about 2000 IU of the long acting FIX polypeptide, (ii) about 59.5 mg sucrose; (iii) about 119 mg nectar a sugar alcohol; (iv) about 19.4 mg L-histamine; and (v) about 0.50 mg polysorbate 20 or polysorbate 80; and (b) the lyophilized powder contained in the first container When combined, the volume is sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, or about 400 IU/ml, respectively. FIX polypeptide; (ii) about 1.2% (w/v) sucrose; (iii) about 2.4% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 25 mM L-histamine And (vi) about 0.01% (w/v) polysorbate 20 or polysorbate 80. The method of claim 120, wherein the medical kit comprises: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 3000 IU of the long acting FIX polypeptide, (ii) about 83.3 mg sucrose; (iii) about 167 mg of mannitol; (iv) about 27.2 mg of L-histamine; and (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and (b) included with the first The lyophilized powder of the container is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 600 IU/ml of the long acting FIX polypeptide; (ii) about 1.7% ( w/v) sucrose; (iii) about 3.3% (w/v) mannitol; (iv) about 55.6 mM NaCl; (v) about 35 mM L-histamine; and (vi) about 0.014% (w/ v) Polysorbate 20 or polysorbate 80. The method of claim 120, wherein the pharmaceutical composition comprises: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 250 IU, about 500 IU, about 1000 IU, or about 2000 IU of the long-acting effect. FIX polypeptide, (ii) about 59.5 mg sucrose; (iii) about 119 mg mannitol; (iv) about 19.4 mg L-histamine; and (v) about 0.50 mg polysorbate 20 or polysorbate 80 ;and (b) comprising, in combination with the lyophilized powder of the first container, a second container having a volume sufficient to produce 0.325% (w/v) NaCl comprising: (i) about 50 IU/ml, about 100 IU, respectively. /ml, about 200 IU/ml or about 400 IU/ml of the long acting FIX polypeptide; (ii) about 11.9 mg/ml sucrose; (iii) about 23.8 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl (v) about 3.88 mg/ml L-histamine; and (vi) about 0.10 mg/ml polysorbate 20 or polysorbate 80. The method of claim 120, wherein the medical kit comprises: (a) a first container comprising a lyophilized powder, wherein the powder comprises (i) about 3000 IU rFIXFc polypeptide, (ii) about 83.3 mg sucrose; Iii) about 167 mg of mannitol; (iv) about 27.2 mg of L-histamine; and (v) about 0.7 mg of polysorbate 20 or polysorbate 80; and (b) contained in the first container The lyophilized powder is combined in a volume sufficient to produce a second container comprising 0.325% (w/v) NaCl of the following solution: (i) about 600 IU/ml rFIXFc polypeptide; (ii) about 16.7 mg/ml sucrose; (iii) about 33.3 mg/ml mannitol; (iv) about 3.25 mg/ml NaCl; (v) about 5.43 mg/ml L-histamine; and (vi) About 0.14 mg/ml polysorbate 20 or polysorbate 80. The method of any one of claims 120 to 124, wherein the first container is a glass vial comprising a rubber stopper. The method of any one of claims 120 to 125, wherein the second container is a syringe body, and wherein the syringe body is accompanied by a plunger. The method of claim 126, the medical kit further comprising a joint for attaching the glass vial to the syringe body. The method of claim 126 or 127, wherein the medical kit further comprises an infusion tube adapted for intravenous infusion with a needle to be attached to the syringe. The method of any one of claims 1 to 128, wherein the human subject does not produce an inhibitor against FIX after administration of the long acting FIX polypeptide. The method of claim 129, wherein the inhibitor is a neutralizing antibody against FIX.